Neurodevelopmental disorders

On-demand webinar

webinar-image

Summary:

Gain a deeper understanding of how impaired neurodevelopment can result in the onset of neurodevelopmental disorders during pregnancy and the early years of childhood in this live interactive digital session.

Neurodeveleopment

Speakers:

Dr. Ki-Jun Yoon, KAIST, South Korea
Gene dosage imbalance of risk factors for neurodevelopmental disorders
Time stamp: 00:07:40

Dr. John Jia En Chua, National University of Singapore, Singapore
The role of FEZ1 in the formation of neuronal networks
Time stamp: 00:35:35

Dr. Noriko Osumi, Tohoku University, Japan
Paternal aging and its effects on the offspring in regard with transgenerational epigenetics
Time Stamp: 1:21:32

Moderator

Dr. Aniko Karpati, Abcam, United Kingdom

Video Transcript

00:00 - 00:17: Hi, thank you for joining the Neurodevelopmental Disorders Session of Spotlight on Neuroscience,
00:17 - 00:22: our month-long virtual conference. We have three wonderful talks for you today
00:22 - 00:29: from experts across the globe. My name is Sian Constantine, and I am the Strategic Marketing
00:29 - 00:35: Manager for Neuroscience at Abcam. Before we begin, I'd like to run through a couple of
00:35 - 00:43: housekeeping notes. All attendees are automatically muted. However, please feel free to submit your
00:43 - 00:49: questions in the Q&A box at the bottom of your screen, and those questions will be addressed
00:49 - 00:56: during the dedicated Q&A session at the end of each talk. If we don't get to your question,
00:56 - 00:59: we will get a response over to you shortly after the event.
01:00 - 01:07: I'm also excited to inform you that Abcam is approved as a provider of continuing education
01:07 - 01:16: programs in the clinical laboratory sciences by the ASCLS PACE program. PACE credits are
01:16 - 01:21: available for this event, and a link to request credit will be sent in the chat box at the
01:21 - 01:30: conclusion of the event. So now I'd like to briefly talk to you about what Abcam does
01:30 - 01:39: in the neurodevelopmental disorders space. So we have a large part of our research that goes
01:39 - 01:47: into neuroscience, and we would like to work to advance the needle in science, in neuroscience
01:47 - 01:54: specifically. And we do that by creating research tools that you can use to speed up your research
01:55 - 02:01: and ultimately to perform experiments better and faster and get data quicker.
02:02 - 02:09: So there are three areas that we're working on at the moment. Neurodevelopment is one big area.
02:11 - 02:16: Neurobiological processes is another, and then the third is neurological diseases.
02:17 - 02:25: And neurological diseases also includes neurodevelopmental diseases. So we have worked
02:26 - 02:34: with multiple companies over the years and academics to make sure we have the right
02:34 - 02:41: research tools for the industry at the right time. A good example of this you can find on our website
02:41 - 02:48: is with the Michael J. Fox Foundation, and we worked very closely with them to develop
02:49 - 02:56: quite a few Parkinson's research tools so that they were the right targets available for researchers
02:56 - 03:02: to use in the laboratory. So if you would like to work with us on making sure that we have the
03:02 - 03:08: right products available, maybe you're in your lab and there's something you really need and
03:08 - 03:14: you can't find and you think, we wish Abcam made this product or this target, please do reach out
03:14 - 03:19: to us at neuroscience.abcam.com and we'd love to hear from you.
03:21 - 03:28: So there are multiple types of research tools that Abcam makes in the neuroscience space.
03:28 - 03:32: We are most well known for antibodies. That's the cornerstone of what we do.
03:33 - 03:42: But we've also utilized the core capabilities that we have to make ELISA kits and proteins
03:42 - 03:49: and peptides that can be used in research too. In addition to that, what's maybe less well-known
03:49 - 03:56: in the neuroscience space is our cellular biochemical assays, but also our cell lines
03:56 - 04:06: and lysates. So we have two very, very interesting iPSC cell lines for glutamatergic neurons
04:06 - 04:14: and skeletal myocytes. And they're both stem cell-derived lines that are very easy to
04:14 - 04:22: differentiate. So that's an area that we're building more and more. So again, we'd love to
04:23 - 04:29: speak to you and hear from you if you're interested in helping us with neuroscience
04:29 - 04:34: products or even interested in purchasing some for your research. And then another area that's
04:35 - 04:41: not very well-known for us in the neuroscience space is our microRNA and immunoassays.
04:42 - 04:47: So again, please, please feel free to look at our website, see what we've got there,
04:47 - 04:52: but also reach out if there's anything you think that we should be doing to help advance the needle
04:52 - 04:58: in science. And finally, I would also like to point out that if we don't have a product that
04:58 - 05:04: you need for your research, and if it's maybe going to take a bit too long for us to build it
05:04 - 05:09: and put it in the catalogue, we do have the customization services where you can
05:10 - 05:13: purchase a customized product for your research.
05:16 - 05:23: So this slide gives you a little flavor of the different types of products we do have for the
05:23 - 05:30: neurodevelopmental disorder space. So here you can see examples of different products we have
05:31 - 05:36: that fit different targets that are specific to neurodevelopmental disorders.
05:37 - 05:42: So, for example, we have got the antibodies in the ELISA kits there,
05:42 - 05:50: but we've also got the human iPSC-derived cells, the conjugates, and proteins and peptides, and so
05:50 - 05:59: on. So without further ado, I would now like to take a moment to introduce the moderator for
05:59 - 06:07: today's event, Dr. Aniko Karpati. Aniko Karpati is the Commercial Strategy Manager for Neuroscience
06:07 - 06:13: at Abcam. She is responsible for the development of Abcam's neuroscience portfolio and partnership
06:13 - 06:19: relationships. In her role, she identifies emerging neuroscience targets and works closely
06:19 - 06:24: with the new product development team to build a relevant and highly validated neuroscience
06:24 - 06:30: portfolio spanning multiple product categories. Before joining the research area strategy team,
06:31 - 06:37: Aniko supported Abcam's customers as a scientific support specialist with a focus on technical
06:37 - 06:45: sales. Aniko obtained her PhD in neuroscience at the Tokyo University in Japan as a doctoral fellow
06:45 - 06:51: of the Japan Society for the Promotion of Science. Her research interests are astrocyte physiology
06:51 - 06:57: and the role of histamine as a neurotransmitter. Applying in vitro and in vivo techniques,
06:57 - 07:01: she unraveled how histamine contributes to astrocyte signaling.
07:02 - 07:06: Thank you. I will now hand the mic over to Aniko.
07:10 - 07:16: Thank you, and welcome to today's session. I'm delighted to also welcome our three experts
07:16 - 07:21: from the field of neurodevelopmental disorders who will be sharing their latest research findings
07:22 - 07:28: in today's session. We already have quite a number of participants in this session, and I really look
07:28 - 07:34: forward to having an engaging session. So, please feel free to drop your questions if you have any
07:34 - 07:41: questions for our speakers, and we can address them after each presentation. With this, I would
07:41 - 07:48: like to introduce our first speaker for today, Dr. Ki-Jun Yoom, who joins us from Korea. He received
07:48 - 07:54: his PhD in developmental genetics at the Pohang University of Science and Technology in Korea.
07:55 - 08:01: In 2018, he established his own lab at KAIST, the Korea Advanced Institute of Science and Technology.
08:02 - 08:08: His lab investigates how neural stem cells are regulated to achieve precise cell division and
08:08 - 08:15: migration, as well as cell fate specification and differentiation. And using both mouse and
08:15 - 08:20: human-induced pluripotent stem cell models, Dr. Yoom's lab focuses on understanding the
08:20 - 08:27: regulation of neural stem cells during neuronal development and in the adult brain. Thank you,
08:27 - 08:31: Ki-Jun, for joining today's event. With this, I will hand over the microphone.
08:33 - 08:39: Yeah, thank you very much for a wonderful introduction, Aniko, and also I'm very
08:39 - 08:46: thankful for the moderators and organizers for these important events. And can you see my slide
08:46 - 08:56: clearly? Okay. So, yeah. So, today I will discuss the gene dosage imbalance of risk factors
08:56 - 09:02: for neurodevelopmental disorders, and using animal models, I will also introduce how we can
09:02 - 09:08: investigate this important topic for the study of neurodevelopmental disorders.
09:10 - 09:15: So, neurodevelopmental disorders are disabilities associated with the functioning of the brain,
09:15 - 09:21: which can be caused by an impaired development of the brain. So, these include
09:21 - 09:26: autism spectrum disorders (ASD), epilepsy, and so on.
09:31 - 09:37: So, copy number variations (CNVs) are alterations of the genome resulting in the abnormal number of copies
09:37 - 09:44: in some sections of the DNA, which might be deleted or duplicated. So, this is a typical example of CNV
09:44 - 09:52: detection by high-density DNA or nucleotide array. So, some regions have more copy number, implying duplication,
09:52 - 09:58: and some regions have less copy number, implying deletion. So, these are risks of the candidate CNVs for the risk of
10:05 - 10:13: schizophrenia. So, multiple CNVs are associated with schizophrenia, and there are sometimes many
10:13 - 10:19: genes and sometimes a few genes inside of these CNVs, and those CNVs have different penetrance
10:19 - 10:29: for the disease occurrence. And 15q11.2 CNVs are risk factors for various neurodevelopmental
10:29 - 10:34: disorders. As described in this table, these are associated with intellectual disabilities
10:34 - 10:39: and autism spectrum disorders and schizophrenia. And this region is relatively short compared to
10:39 - 10:48: other well-known CNVs, including four genes: GCP5, CYFIP1, NIPA1, and NIPA2. So, my initial question
10:48 - 10:54: is, what are the cellular molecular deficits associated with 15q11.2 CNVs? So, I utilized
10:54 - 11:02: induced pluripotent stem cells harboring the 15q11.2 microdeletions and differentiated those iPSCs
11:02 - 11:08: to neural precursor cells, which tend to form a neural rosette during two-dimensional
11:08 - 11:14: differentiation. As you see here, in healthy controls, we can see nicely polarized neural
11:14 - 11:23: rosettes and also apical junction markers and tight junction markers, like PKC around there inside in the
11:23 - 11:29: center of the neural rosette. However, the neural rosette harboring the 15q11.2 microdeletions
11:29 - 11:34: has impaired polarity of the neural rosette and also the tight junction structures,
11:34 - 11:41: which can be rescued by the overexpression of one of these CNV genes, CYFIP1.
11:42 - 11:47: So, we suggest that CYFIP1 is responsible for the maintenance of apical polarity
11:47 - 11:54: during neural development. This work was published a long time ago, and we further analyzed
11:55 - 12:01: the signaling pathways and also associated cortical malformations with CYFIP1 loss of function.
12:02 - 12:09: So, today's topic, I changed the topic a little bit and focused on the gene dosage effect
12:09 - 12:16: of these risk factors. So, neuronal homeostasis is crucial for proper brain function. So,
12:16 - 12:23: in many cases, both gain of function and loss of function by gene deletion or gene duplication
12:23 - 12:29: affect the synaptic output overall, which eventually leads to the development of psychiatric disorders,
12:29 - 12:38: such as ASD or schizophrenia. So, this is a list of the risk factors that contain
12:38 - 12:45: the gene dosage effect affecting the neurodevelopmental disorders. And as I said, 15q11.2
12:45 - 12:51: CNVs are associated with diametric risk for schizophrenia and autism spectrum disorders.
12:51 - 12:58: These 15q11.2 deletions have been identified as one of the three most frequent CNV risk
12:58 - 13:04: factors for schizophrenia, whereas the duplication of the same region is associated with ASD risk.
13:05 - 13:13: Interestingly, structural MRI studies showed that these duplications and deletions also
13:13 - 13:19: lead to structural reformations, which shows a reciprocal effect. Some regions are increased in
13:19 - 13:25: the duplication, but some regions are decreased. The size of the brain structure is decreased
13:25 - 13:34: with the deletion. All these results suggest that 15q11.2 CNVs are prominent dosage-sensitive
13:34 - 13:39: genetic risk factors for neurodevelopmental disorders. However, it remains unclear how
13:39 - 13:44: different dosages of this region may contribute to the etiology of underlying neurodevelopmental
13:44 - 13:52: disorders. So, four genes located in the 15q11.2 region, but in this study, we focused on one
13:52 - 14:00: candidate gene, CYFIP1, for the mechanisms. CYFIP1 is known as a translational repressor at the synapse.
14:01 - 14:08: It acts as a non-canonical IRES binding protein and suppresses the initiation
14:08 - 14:17: of the synaptic translation of certain target mRNAs. So, my question is, are there any behavioral
14:17 - 14:23: abnormalities caused by altered CYFIP1 doses? And which mRNAs are the targets of CYFIP1-mediated
14:23 - 14:29: translation regulation? And can these behavioral abnormalities be rescued by the mechanism-based
14:29 - 14:34: pharmacological intervention, if we can find some kind of mechanism? So, to do that,
14:34 - 14:51: we generated a model system that is a loss-of-function model by conditional knockout of CYFIP1,
14:51 - 14:56: which deletes certain axons, including critical axons, by the nesting tree. So, it deletes the
14:56 - 15:03: CYFIP1 protein in the entire nervous system, and we confirmed the deletion of the CYFIP1 protein
15:03 - 15:13: in the brain. We also generated a conditional overexpression system. We inserted the CYFIP1 cDNA
15:14 - 15:19: into the ROSA26 locus, so we could delete the CYFIP1 proteins in the entire nervous system.
15:19 - 15:29: We confirmed that these overexpression models exhibit an expression level of 1.5-fold or 2-fold
15:30 - 15:35: increase of CYFIP1 dosage in these animal models. And also, as I mentioned earlier, we applied
15:35 - 15:42: the patient-derived iPSC model harboring 15q11.2 deletion, and differentiated those iPSCs
15:42 - 15:49: to the forebrain neurons. We also confirmed that the CYFIP1 level was reduced in these in vitro systems.
15:51 - 15:56: So, my first question is, what are the behavioral consequences caused by altered CYFIP1 dosages?
15:56 - 16:03: So, I applied a battery of behavioral tests that are associated with schizophrenia or autism symptoms.
16:03 - 16:08: First of all, the CYFIP1 conditional knockout mouse shows impaired social interaction,
16:08 - 16:13: which is revealed by the three-chamber test. In stage one, we interacted the mouse with a stranger
16:13 - 16:20: mouse versus an empty cage. In the second stage, we interact this experimental animal with a normal mouse
16:26 - 16:32: and a familiar mouse. As a result, the CYFIP1 knockout mouse doesn't show a defect in the social approach
16:34 - 16:38: in the first session, but however, it shows a decreased recognition of social novelty, suggesting that
16:45 - 16:52: these mice have a mild social interaction impairment. The knockout also shows impaired preference
17:00 - 17:05: inhibition, which is the inability to pick out unnecessary information,
17:05 - 17:11: which is associated with the schizophrenia symptom in human patients. The knockout mouse shows
17:11 - 17:15: decreased preference inhibition in the entire session and also increased subtle response,
17:15 - 17:21: suggesting they have a problem with sensory-motor gain.
17:21 - 17:27: The knockout mice also showed amphetamine-induced hyperactivity. Compared to
17:27 - 17:28: the wild type, the knockout mouse shows more activity in the open-chamber test,
17:28 - 17:33: which is automatically assessed by the beam-break numbers and is increased in the knockout mouse.
17:34 - 17:40: Typically, many schizophrenia model mice show behavioral despair. So, we tested
17:42 - 17:49: depression behavior with the knockout mice using the tail suspension test and forced swim tests.
17:49 - 17:55: In both cases, we see increased behavioral despair with the deletion of the CYFIP1 mouse models.
17:55 - 18:03: On the other hand, I also analyzed multiple behavioral tests in the CYFIP1 overexpression mouse, and this
18:03 - 18:08: overexpression mouse shows more severe social impairment. So, they show significant reduction of the
18:08 - 18:14: social approach as well as social novelty. Interestingly, this overexpression mouse displays very strong
18:22 - 18:28: repetitive behavior, obsessive behavior. For example, in the marble-burying test that I performed,
18:28 - 18:37: the wild type does not hide the marble inside the bedding that much,
18:37 - 18:43: but the overexpression mice are very dedicated to hiding marbles inside of the bedding, which suggests
18:43 - 18:51: that they exhibit typical obsessive behavior in this test. The number of marbles buried
18:51 - 18:57: during the session is significantly increased, and they have very extensive digging periods during
18:58 - 19:08: these sessions. The overexpression mice also show impaired maternal behaviors,
19:08 - 19:15: including nest-building behavior, which is very natural for the wild type mouse. However,
19:15 - 19:22: after overnight sessions, the overexpression mice do not show the behavior of nest-building,
19:22 - 19:30: which is assessed by weight and nesting score.
19:29 - 19:37: I also performed a pup retrieval test. For this test, I placed the young pups, the neonatal
19:37 - 19:44: pups in three corners of the cage and put the animal in the center. The wild type mom
19:44 - 19:47: collected the pups diligently.
19:51 - 19:58: However, the overexpression mice showed extensive digging and did not attempt to collect the pups
20:00 - 20:05: to a certain location in the cage. Here you can appreciate how the wild type mice
20:09 - 20:12: collected all three pups in the corner,
20:12 - 20:15: while the overexpression mice did not care for the pups at all.
20:19 - 20:23: These results suggest a spectrum of behavior impairment with the knockout mouse
20:23 - 20:26: and overexpression mouse. Some of the behaviors in the knockout mouse
20:30 - 20:33: are associated with schizophrenia symptoms,
20:33 - 20:35: and also, the overexpression mice,
20:37 - 20:42: although they share some behavior impairment,
20:42 - 20:46: the overexpression mice show typical ASD-associated
20:46 - 20:48: behaviors, such as repetitive behavior
20:48 - 20:50: and impairment of maternal behaviors.
20:52 - 20:55: So, my next question is, what is the molecular mechanism
20:55 - 20:57: underlying this behavioral alteration?
20:57 - 21:00: To tackle that question,
21:00 - 21:05: I first investigated which mRNAs are targets
21:05 - 21:08: of CYFIP1-mediated translation regulation.
21:08 - 21:12: To do that, I applied RIP sequencing technology.
21:12 - 21:14: We pulled down the CYFIP1 protein
21:14 - 21:17: from the adult hippocampus,
21:17 - 21:20: and also sequenced the associated
21:20 - 21:23: interacting RNA species by high-throughput sequencing.
21:25 - 21:27: As a result, we revealed that CYFIP1
21:27 - 21:30: interacts with numerous mRNA candidates,
21:30 - 21:34: which are related to post-synaptic density
21:34 - 21:37: or dendritic spine formation,
21:37 - 21:38: especially components of NMDA receptors.
21:40 - 21:41: This is an example.
21:41 - 21:44: Famous autism-associated genes,
21:44 - 21:48: such as SHANK1, SHANK2, and SHANK3,
21:48 - 21:50: are CYFIP1-interacting proteins.
21:51 - 21:55: We confirmed that many post-synaptic density proteins
21:55 - 21:58: and NMDA receptor complex-associated mRNAs
21:58 - 22:01: interact with the CYFIP1 protein
22:01 - 22:03: in the adult mouse hippocampus.
22:03 - 22:07: This was validated by QPCR experiments,
22:07 - 22:11: where we saw that the enrichment of GRIN2A,
22:11 - 22:13: NMDA receptor subunits,
22:13 - 22:15: as well as post-synaptic proteins,
22:15 - 22:19: such as SHANK and PSD95, interacts with the CYFIP1 proteins.
22:19 - 22:21: We also confirmed that interaction
22:21 - 22:26: with human cortical lysate by QPCR experiments, as shown here.
22:29 - 22:33: So I wondered about the effect of CYFIP1 binding on these target mRNAs.
22:33 - 22:34: So I applied translation monitoring tools.
22:34 - 22:39: I used puromycin labeling on hippocampal lysates.
22:42 - 22:45: After ribosome puromycin labeling,
22:45 - 22:48: I applied puromycin to the lysate,
22:48 - 22:50: releasing the nascent peptides
22:50 - 22:53: to the supernatant.
22:53 - 22:56: I collected those released nascent peptides
22:56 - 23:01: using streptavidin beads and performed western blotting.
23:05 - 23:08: Overall, the total translation efficiency
23:08 - 23:13: is not significantly altered by the deletion of CYFIP1,
23:14 - 23:15: as shown here.
23:15 - 23:17: However, the specific target proteins,
23:17 - 23:18: such as NMDA receptor subunits
23:18 - 23:21: or postsynaptic proteins,
23:22 - 23:23: their translation rates,
23:23 - 23:26: and the levels of nascent peptides are increased
23:26 - 23:29: with the deletion of CYFIP1.
23:29 - 23:36: Interestingly, the same target proteins show reduced expression,
23:36 - 23:37: and the translation rates for the same target species
23:37 - 23:41: are reduced in the overexpression of CYFIP1 in the mouse models.
23:43 - 23:52: Moreover, I examined the levels of the proteins in the synapse
23:52 - 23:54: in the knockout model and overexpression model,
23:54 - 23:55: and the resultant protein levels in the synaptosome
23:55 - 24:05: from knockout and overexpression models also showed reciprocal interactions.
24:06 - 24:08: For example, NMDA receptor subunits,
24:08 - 24:10: their protein levels are increased,
24:10 - 24:15: while in the overexpression model, the same targets are reduced.
24:17 - 24:25: Also, human neurons with 15q11.2 deletion
24:25 - 24:28: also show exaggerated expression,
24:28 - 24:31: and protein expression of the NMDA receptor subunits,
24:31 - 24:33: suggesting that this is a common
24:33 - 24:37: shared property between our mouse and human models.
24:39 - 24:40: Functionally,
24:40 - 24:43: I examined the NMDA receptor functions
24:43 - 24:46: in both the wild-type and knockout mice,
24:46 - 24:51: and revealed that NMDA receptor-mediated currents
24:52 - 24:54: assessed by the NMDA/AMPA ratio
24:54 - 24:57: through electrophysiological recordings
24:57 - 25:04: are also elevated in the knockout model,
25:04 - 25:07: while decreased in the overexpression models.
25:08 - 25:10: NMDA receptor downstream signaling
25:10 - 25:12: is also exaggerated in the knockout models,
25:12 - 25:15: but those pathways are reduced in the overexpression model.
25:16 - 25:20: Lastly, I would like to assert that,
25:20 - 25:22: can this behavioral abnormality
25:22 - 25:24: be rescued by mechanism-based
25:24 - 25:26: pharmacological intervention?
25:26 - 25:30: We observed alterations of NMDA receptor signaling,
25:30 - 25:33: and by modulating NMDA receptor signaling
25:33 - 25:34: in these animal models,
25:34 - 25:36: can we rescue the behavioral phenotypes?
25:36 - 25:40: So, I applied a mild NMDA receptor antagonist
25:41 - 25:43: to the knockout model
25:43 - 25:47: to suppress the elevated NMDA receptor signaling.
25:47 - 25:50: After treatment with memantine,
25:50 - 25:53: depression behavior is nicely suppressed
25:53 - 25:54: in the knockout model.
25:54 - 25:58: So, in the knockout model, as mentioned,
25:58 - 26:00: they show increased behavioral despair,
26:00 - 26:02: and this is nicely suppressed
26:02 - 26:05: by the treatment with memantine.
26:05 - 26:08: Furthermore, the amphetamine-mediated hyperactivity
26:08 - 26:13: is one of the dramatic phenotypes in the knockout model.
26:13 - 26:14: This is also well suppressed
26:14 - 26:17: by the treatment of memantine in the knockout model.
26:17 - 26:21: suggesting that suppressing the NMDA receptor activity
26:21 - 26:24: may contribute to some of the behavioral phenotype
26:24 - 26:26: of the knockout mouse models.
26:27 - 26:28: On the other hand,
26:28 - 26:31: I tried to enhance the NMDA receptor signaling
26:31 - 26:35: by treatment with D-cycloserine in the overexpression mice.
26:35 - 26:37: And after the treatment with D-cycloserine,
26:37 - 26:42: the social impairment of the overexpression mice
26:42 - 26:43: is nicely covered.
26:43 - 26:45: So, as you see here,
26:45 - 26:49: the overexpression mouse shows reduced social interaction
26:49 - 26:51: with the stranger mouse,
26:51 - 26:55: and this reduction can be well ameliorated
26:55 - 26:57: by the treatment with D-cycloserine.
26:57 - 27:00: And also, social normative recognition
27:00 - 27:05: is impaired in the overexpression mice without treatment.
27:05 - 27:06: By rapid treatment,
27:06 - 27:09: this behavior is improved
27:09 - 27:12: by enhancing the NMDA receptor signaling pathway.
27:13 - 27:14: So, here is a summarization.
27:14 - 27:16: As depicted in this diagram,
27:16 - 27:18: both loss-of-function and gain-of-function
27:18 - 27:20: lead to the imbalance of the neuronal signaling pathway,
27:20 - 27:22: such as the NMDA receptor pathway.
27:22 - 27:25: So, in this case, the gene dosage imbalance
27:25 - 27:27: leads to the bidirectional impairment
27:27 - 27:29: of co-synaptic signaling.
27:29 - 27:31: In the case of the CYFIP1,
27:31 - 27:34: that would be the NMDA receptor signaling.
27:34 - 27:36: And both loss-of- and gain-of-function
27:36 - 27:39: lead to divergent behavioral abnormalities
27:39 - 27:42: related to schizophrenia and autism.
27:43 - 27:45: And imbalanced CYFIP1 doses
27:45 - 27:48: lead to the changes of NMDA receptor-associated
27:48 - 27:53: complex proteins via dysregulated translation.
27:53 - 27:55: So, CYFIP1 inhibits the translation
27:55 - 28:00: of certain specific target mRNAs at the synapse,
28:00 - 28:04: and these are a cause of the reciprocal alteration
28:04 - 28:07: of the protein expression at the synapse.
28:07 - 28:10: So, some of the behavioral abnormalities can be rescued,
28:10 - 28:11: not all of the behavior,
28:11 - 28:13: but some of the behavior can be rescued
28:13 - 28:16: by bidirectional pharmacological balancing
28:16 - 28:17: of the NMDA receptor signaling.
28:17 - 28:20: So, these works are recently published
28:20 - 28:23: in Biological Psychiatry this year.
28:23 - 28:27: So, if you want to see more details about the data,
28:27 - 28:29: please find these papers.
28:30 - 28:35: Most of the work I present here,
28:35 - 28:39: actually, is done in my post-doc lab,
28:39 - 28:41: University of Pennsylvania,
28:41 - 28:45: under the supervision of Dr. Hongjun Song and Gori Ming.
28:45 - 28:48: I really appreciate their guidance.
28:48 - 28:51: And many people are involved in this work,
28:51 - 28:54: and behavior at Johns Hopkins Behavioral Corp
28:54 - 28:58: and the collaborator has all the projects.
28:58 - 29:01: And recently, I set up my own lab in South Korea,
29:01 - 29:04: KAIST, Korea Advanced Institute of Science and Technology.
29:04 - 29:08: So, please visit my website for the information.
29:08 - 29:10: Yeah, thank you very much for listening,
29:10 - 29:12: and I would be happy to take any questions.
29:15 - 29:18: Thank you, Ki-Jun, for your presentation.
29:18 - 29:22: It was very interesting to hear about the role of CYFIP1
29:22 - 29:25: in neurodevelopmental disorders,
29:25 - 29:28: and yes, we are happy to now take questions
29:28 - 29:30: from the audience.
29:30 - 29:32: Please drop them in the chat box.
29:32 - 29:34: And there's already a first question
29:34 - 29:35: that I would like to ask.
29:36 - 29:41: The question is related to the nest-building behavior test.
29:41 - 29:45: And the question is whether the pups are wild-type mice
29:45 - 29:48: or whether they have the same genotype as the mother.
29:50 - 29:52: Yes, I mean, that's a very interesting question.
29:52 - 29:54: So, in this particular test,
29:54 - 29:57: we only used the wild-type pups,
29:57 - 30:01: but we also observed a mild disturbance
30:01 - 30:06: of the ultrasonic vocalizations of the mutant pups,
30:08 - 30:11: but I didn't present that here.
30:11 - 30:15: But I think it's also an interesting future direction
30:15 - 30:20: to investigate the interaction between mom and pups
30:20 - 30:22: with the different genotypes,
30:22 - 30:25: but we don't have plenty of data
30:25 - 30:27: to conclude anything from that,
30:27 - 30:30: but we observed some mild disturbances
30:30 - 30:33: of the vocalizations from the knockout,
30:33 - 30:37: CYFIP1 knockout pups to wild-type mothers.
30:37 - 30:38: Okay.
30:39 - 30:44: And another question is about the interaction of CYFIP1
30:46 - 30:47: with other targets that are not related
30:47 - 30:50: to the NMDA receptor complex.
30:50 - 30:52: So, are there any other mechanisms
30:52 - 30:56: that would explain the behavioral abnormalities
30:56 - 30:57: that you have observed?
30:58 - 31:02: Yes, I think there are numerous other targets,
31:02 - 31:07: not only just for the NMDA receptor complex-associated genes.
31:07 - 31:12: So, I think we examined the number of dendritic spines
31:14 - 31:16: or the morphology of spines with the knockouts,
31:16 - 31:19: and we observed severe alterations as well.
31:19 - 31:22: So, probably the other mRNAs,
31:22 - 31:24: which contribute to the formation of synapses
31:24 - 31:29: or the structural aspects of the neurons
31:29 - 31:32: also do something here.
31:32 - 31:36: But in my previous study, I mean, in the present study,
31:36 - 31:38: I only focused on the reciprocal
31:38 - 31:43: or diametric signaling in this dosage effect.
31:43 - 31:46: So, we are currently investigating this aspect
31:46 - 31:49: of the structural alteration of the synapse
31:49 - 31:53: or activity-dependent protein translation or other things.
31:53 - 31:57: Maybe other targets also actively participate
31:57 - 31:58: in these processes.
31:59 - 32:02: And then you just mentioned that the number and morphology
32:02 - 32:06: of the synapses in the mouse models are altered.
32:06 - 32:08: Could you elaborate a bit more on this?
32:10 - 32:14: Yes, I mean, we observed a reduction
32:14 - 32:16: in the dendritic spine number
32:17 - 32:21: and a reduction of the mature dendritic spines
32:21 - 32:22: with the CYFIP1 deletion.
32:22 - 32:26: And also, we see a mild increase of the,
32:26 - 32:27: I don't have data here,
32:27 - 32:32: but also a mild increase in the dendritic spine maturation
32:32 - 32:34: by the overexpression of CYFIP1.
32:34 - 32:38: So, I think that is also related to the actin remodeling
32:38 - 32:40: because CYFIP1 is also interacting
32:40 - 32:42: with the actin remodeling complexes
32:42 - 32:46: and modulates their activity
32:46 - 32:49: of the actin cytoskeleton rearrangement,
32:49 - 32:52: which is very crucial for spine formation
32:52 - 32:54: and the physiology of dendritic spines.
32:55 - 32:59: That's actually the current ongoing direction of my laboratory.
32:59 - 33:01: Okay.
33:01 - 33:02: Another question that came through
33:02 - 33:06: is regarding the frequency of mutations,
33:06 - 33:10: CYFIP1 mutations in patients with neuropsychiatric disorders.
33:12 - 33:15: So, do you know anything about the frequency
33:15 - 33:17: of mutations occurring?
33:19 - 33:21: Yeah, I think here,
33:21 - 33:25: this is the frequency of the mutation
33:25 - 33:30: with the P50-Q1 deletion in the schizophrenic patients,
33:30 - 33:34: which is actually kind of not super dominating,
33:34 - 33:38: but it is frequent for CNVs,
33:38 - 33:42: we're within the top four CNVs
33:42 - 33:44: associated with schizophrenia human genetics.
33:46 - 33:50: And there are also CYFIP1 CNVs,
33:50 - 33:55: CYFIP1 single nucleotide variants
33:55 - 33:58: associated with schizophrenic patients.
33:58 - 34:00: Those are reported in the human genetic studies.
34:00 - 34:02: I don't remember the exact frequencies,
34:02 - 34:07: but frequencies of CNVs are presented in this table.
34:07 - 34:08: Okay.
34:09 - 34:12: Another question is around patient stratification.
34:13 - 34:14: So the question is,
34:14 - 34:16: how to clinically distinguish the patient
34:16 - 34:19: on the level of NMDA signaling?
34:21 - 34:24: That's very, yeah, a very difficult question.
34:24 - 34:29: So, yeah, ongoing the NMDA receptor signaling,
34:31 - 34:33: the strengths of NMDA signaling
34:33 - 34:36: or other aspects of NMDA signaling in patients
34:36 - 34:41: is hard to probably maybe rebuild by the PET imaging,
34:41 - 34:44: but I don't have much information on it.
34:44 - 34:48: Yeah, but we only get some information
34:48 - 34:50: from the genetic studies,
34:50 - 34:52: what kind of mutations are associated
34:52 - 34:57: and which type of CNV or SNV is related to these disorders.
34:58 - 35:01: And we start from there using animal models.
35:01 - 35:06: So I also wonder how the actual patients
35:06 - 35:10: with the CYFIP1 mutations show the NMDA receptor,
35:10 - 35:11: alters NMDA receptor signaling,
35:11 - 35:15: probably that's an interesting topic in the future.
35:15 - 35:16: Okay.
35:16 - 35:16: Yeah, thanks.
35:16 - 35:18: Yeah, thank you.
35:20 - 35:24: Yeah, thank you for answering all the questions.
35:24 - 35:26: It was a great talk.
35:26 - 35:27: Thank you so much.
35:27 - 35:31: And I will now move on to the second speaker.
35:31 - 35:36: And our second speaker today is Dr. John Jia En Chua.
35:37 - 35:39: And he's an assistant professor
35:39 - 35:42: in the Department of Physiology and Healthy Longevity
35:42 - 35:44: Translational Research Program
35:44 - 35:47: at the Yong Loo Lin School of Medicine
35:47 - 35:49: at the National University of Singapore.
35:49 - 35:52: He's also a joint principal investigator
35:52 - 35:54: of the Institute of Molecular and Cell Biology
35:54 - 35:56: and principal investigator
35:56 - 35:59: at the Institute for Health Innovation and Technology.
36:00 - 36:04: And John's group combines molecular and biochemical imaging
36:04 - 36:07: but also omics as well as human stem cell
36:07 - 36:10: and model organism technologies to elucidate proteins
36:10 - 36:13: and biological pathways involved
36:13 - 36:15: in neuronal network formation during brain development
36:15 - 36:18: as well as in neurodegenerative conditions.
36:18 - 36:22: I'm now delighted to hand over to John for his presentation.
36:23 - 36:26: Yeah, thanks, Aniko, for the kind introduction
36:26 - 36:29: and I hope you can hear me and I hope you can see the slide.
36:29 - 36:30: Yes.
36:30 - 36:31: Yeah, thanks.
36:31 - 36:34: So it's a pleasure to be, oh, what's going on here?
36:36 - 36:39: Okay, it's a pleasure to be able to share our work
36:39 - 36:40: with this audience.
36:40 - 36:45: And what I would like to try to do is to show you
36:47 - 36:50: how fast time through his involvement
36:51 - 36:53: in the formation of neuronal networks
36:53 - 36:57: can potentially contribute to neurodegenerative disorders.
36:57 - 36:59: In fact, there have been some associations
36:59 - 37:03: of fast time mutations to neuropsychiatric disorders.
37:03 - 37:05: So I think for this audience,
37:05 - 37:10: I don't have to reiterate the idea that brain function,
37:10 - 37:14: normal brain function in particular is intimately related
37:14 - 37:18: to the formation of normal neuronal networks.
37:18 - 37:22: And in the brain, there are local neuronal networks
37:22 - 37:25: or local circuits that are formed
37:25 - 37:27: within individual brain functional regions,
37:27 - 37:30: but also there are long-range networks
37:30 - 37:33: that are formed between distinct regions
37:33 - 37:36: of the brain itself that's important for coordinating,
37:36 - 37:40: for instance, emotion or even thoughts or even memory.
37:40 - 37:42: And I think we're also familiar with the idea
37:42 - 37:45: that neuronal networks are dependent
37:45 - 37:47: on information transfer.
37:47 - 37:51: And this information transfer occurs through synapses
37:51 - 37:56: through which the neurons are connected.
37:56 - 37:58: And for a number of years,
37:58 - 38:00: we have been involved in identifying
38:00 - 38:05: the composition of synapses and identify,
38:07 - 38:11: how the proteins interact with each other
38:11 - 38:16: in order to conduct signaling to transfer information
38:16 - 38:19: from one neuron to another.
38:20 - 38:25: Now, it's probably not too foreign an idea
38:27 - 38:32: that neuronal networks do not occur spontaneously.
38:32 - 38:33: And I think many of us are aware
38:33 - 38:35: that when the brain develops,
38:35 - 38:40: it has to undergo a multi-step process,
38:41 - 38:43: starting with neurogenesis.
38:43 - 38:46: And once immature neurons are formed,
38:47 - 38:48: they have to undergo processes
38:48 - 38:50: that allow them to extend to,
38:50 - 38:53: to reach out to their target neurons
38:53 - 38:58: in other regions of the locus circuit,
38:58 - 39:02: or also importantly to other regions of the brain itself.
39:02 - 39:04: So I'm going to focus more on
39:06 - 39:09: how immature neurons start to produce neurites and to form synapses
39:09 - 39:13: after they reach their designated connections.
39:13 - 39:20: So essentially, as you can see in the cartoon on the right,
39:20 - 39:23: immature neurons have to,
39:23 - 39:27: as they try to form neuronal networks themselves,
39:28 - 39:30: emanate axons and dendrites,
39:31 - 39:33: not just lengthen,
39:33 - 39:37: but they also branch as they migrate
39:37 - 39:40: through the use of various guidance cues
39:40 - 39:43: towards their target neurons in the brain itself.
39:43 - 39:47: And once upon reaching their targets,
39:47 - 39:51: the neurons then form synaptic connections with each other.
39:51 - 39:56: And this initiates the formation of neuronal networks
39:56 - 39:58: in the brain itself
39:58 - 40:00: that is so essential for our brain to function.
40:00 - 40:06: Now to give an idea of how challenging this is.
40:06 - 40:27: I show you on the left. This is just a real roadmap of the major, meaning the high-speed connections in Europe itself. And it doesn't even include the regional lines, not local lines, that connect people from one place to another.
40:28 - 40:44: Now if you consider each city in a point as a neuron, and each railroad line as an axon or dendrite that connects these connections, you probably have a better idea of how resource intensive and how elaborate
40:44 - 40:48: And how prolonged it takes for these networks to form, and it's essentially the same in the development of the human brain itself. So in order for neurons to start from these immature neuronal branching to ultimately forming the neuronal networks
41:12 - 41:16: in the even the young brain itself.
41:16 - 41:31: A lot of logistical demands and a lot of coordination is actually involved in order to obtain a normally functioning neuronal network in the normal brain.
41:32 - 41:54: With this in mind, I think we can appreciate this better, that even though the brain is essentially more or less formed at the stage of the optimization of these networks, as in the case of railway networks continuous optimization takes place over a long span of time.
41:55 - 42:12: In the case of the human brain, it is known that it takes up to about two decades for the brain to optimize the connections that were previously established during the early stages of neuronal development itself.
42:12 - 42:39: So it's actually not too surprising that any perturbations or any abnormalities that affect early brain development can ultimately result in the onset of neuropsychiatric disorders such as ASD, ADHD, and even schizophrenia, that are known to have strong neurodevelopmental origins.
42:39 - 42:42: So, I talked a bit about the daunting logistics of assembling neural networks. And I'm going to focus on one aspect of logistics which is in principle related to the idea that neurons have to extend processes, axons, and dendrites over vast distances,
43:04 - 43:19: by finding them, for instance, that the surface area of neurons themselves expands at a rate of 20% per day, which is actually quite impressive considering the size of the neurons themselves.
43:20 - 43:33: and in order to support such rapid growth, you essentially need to bring new biological materials from the cell body, where most synthesis occurs to sites of growth.
43:33 - 43:36: And as the neurons start to form synapses with each other,
43:36 - 44:02: you also need to carry synaptic proteins in order to allow synaptic formation to occur.
44:02 - 44:21: There are essentially two classes of transport, if one were to generalize, one will be the anterograde or forward transport that brings raw materials from the cell body to the growth sites, or synaptogenesis itself.
44:21 - 44:41: And then there's also the retrograde transport that brings waste, if you like, from the distal sites of the neurites back to the cell body. Of course, it's also involved in new synaptic neurosynaptic soma signaling, which I'm not going to talk about today.
44:41 - 44:56: But I’m going to focus more on the anterograde or forward transport that brings newly synthesized materials from the cell body to growing dendritic tips and sites of neuro synaptic genesis. And a major class of proteins, or so-called motor proteins that drive
44:56 - 45:15: this effort are our kinesins, which in conjunction with adaptor proteins, allow cargoes to be transported from one region, meaning the cell body to areas where they are needed.
45:15 - 45:33: So I'm going to focus a bit more on adaptors today and to try to demonstrate how important it is for adaptor proteins such as fast on the cell
45:16 - 45:37: in the recognition of cargoes, but also in the importance of adaptors themselves in their versatility by acting as signaling hubs to coordinate various signaling mechanisms that are required for neuronal development and the development of neuronal networks.
45:37 - 45:55: So, just to briefly summarize, you know, transport itself, especially anterograde transport itself is important for neurite outgrowth and branching, this I've mentioned before, synaptogenesis, and as the neurons start to form networks, also synaptic
45:55 - 46:04: maintenance, remodeling, turnover, and for the modulation of gene expression.
46:04 - 46:15: So a number of years ago we stumbled upon this protein. So, as I mentioned, we were very interested in synaptic function and synaptic protein interactions, so we performed a large screen.
46:15 - 46:32: And we found a protein called FEZ1, that binds to a very important presynaptic protein known as syntaxin 1, and many of you might know that syntaxin 1 itself is essential for synaptic
46:33 - 46:44: And without going further into that, one of the things that we first tried to identify was, you know, because we are aware that this killer transport is involved.
46:45 - 46:52: Most of the time, it's an efficient way of transporting proteins, and because of the scale of nature.
46:52 - 47:09: The idea that you could group particular cargoes to transport similar components at the same time, which is economically beneficial in terms of energy for neurons is likely to happen.
47:09 - 47:22: So, we started to look at what additional protein cargoes might be present on FEZ1 transport vesicles, and to make a long story short, a lot of these proteins are related to synaptic transmission.
47:22 - 47:28: And at least some of these proteins are also known to be involved in neuronal development itself.
47:28 - 47:38: And what we showed previously was that if you take out FEZ1 function in C. elegans, which were downregulated on 76.
47:38 - 47:44: You start to see external aggregates, which is a hallmark of impaired neuroin transport.
47:44 - 48:04: And as a result of that, synapses are reduced or lost or disorganized. As you can see in the middle panel over here, and we managed to show that such abnormalities can be observed in transgenic mouse models,
48:04 - 48:23: and you can see that aggregation of FEZ1 in the three-times DGT mice, for instance, is very prominent at two years of age, and you can start to see the progressive accumulation of FEZ1 aggregates in these transgenic animals as AD progresses in these animals.
48:23 - 48:39: And what was intriguing for me, at least at the time, was that the very early reports of FEZ1 function, particularly in C. elegans and Drosophila, were related to its role in development.
48:39 - 49:00: So, for instance, in the Blümel and Roberts paper, they showed that loss of FEZ1 function results in external developmental defects, even bundling defects.
49:00 - 49:15: And in this very interesting paper from Corona and coworkers, they showed that if you try to induce neurogenesis in a C. elegans model, the increase in FEZ1 expression is required in order for neurites to emerge.
49:15 - 49:30: And what was interesting was a while ago, from data from Alan Solomon's lab, that it seems that NPR1 by interaction with FEZ1 is required for the development of the neural tube itself.
49:30 - 49:43: So we started asking ourselves, you know, is it possible, and it definitely seems to be the case that FEZ1 could have a greater role to play in neuronal development that we're not aware of.
49:43 - 49:49: And this is the case because if you look at human developmental disorders, FEZ1 mutations, copy number variations, and expression level changes have been linked to schizophrenia, and to a certain extent, ASD,
49:49 - 50:11: and at least in the animal model have been associated with ADHD phenotypes. But even more interestingly, we stumbled upon the involvement
50:11 - 50:24: of FEZ1 deletion in a very rare condition known as Jacobson syndrome, where there are regional deletions of the terminal of chromosome 11q.
50:25 - 50:41: And what's interesting about these patients is that they show behavioral and cognitive defects representative of ADHD, ASD, and schizophrenia, and very rarely bipolar disorders as well.
50:41 - 50:59: And as I mentioned, I probably didn't mention earlier, but you know in the early papers, there was evidence that on 76 mutations of FASD mutations in *C. elegans* confirmed movement disorders in this model organism.
50:59 - 51:11: And it's interesting that these patients, the Jacobson syndrome patients, also exhibit psychomotor impairment, including the loss of gross and fine motor function.
51:11 - 51:18: So, we started to ask ourselves, what is the potential role of FEZ1 in early brain development?
51:18 - 51:25: And how do these abnormalities contribute to neurodevelopmental disorders?
51:25 - 51:32: And the firs thing we did was to go to our primary neuronal cultures, which we generally routinely use in the lab.
51:32 - 51:39: And this is a very simple expression experiment with these medical cultures that, you know, many labs in the world use.
51:39 - 51:49: And if you grow the neurons and you check the levels over the course of development, you see that FEZ1 expression progressively increases as the neurons mature.
51:49 - 51:59: And this tells us that, you know, that's developmentally regulated expression of the protein, which supports our hypothesis.
51:59 - 52:07: And what we decided to do later is to start to knock down FEZ1 expression in these cultures.
52:07 - 52:14: And you can see from the data presented here that once neurons are depleted of FEZ1 function.
52:14 - 52:26: You can see that the effect on axon length, be it total or be on the longest axon branch itself is to inhibit its growth.
52:26 - 52:30: And also, if you look at the number of branch points,
52:30 - 52:39: FEZ1 knockout neurons exhibit a significantly lower number of branch points as compared to control or wild type uninfected neurons themselves.
52:39 - 52:48: So the effect on neuronal growth is not limited to axons. If you look at dendrites as well, you can also see that when you conduct a Sholl analysis.
52:48 - 53:03: FEZ1 knockout neurons have significant and dramatic developmental impairment, and if you see this Sholl analysis or area under the graph, you can see that the impairment in FEZ1 knockout neurons is quite significant.
53:03 - 53:13: So, I mentioned that we did a large screen for protein-protein interactions, and actually one of the screens revealed something very interesting.
53:13 - 53:29: And that's the interaction between FEZ1 itself with CRMP-1. And some of you might know of this as an effector in the Semaphorin 3A guidance cue signaling pathway; I’ll come to this a little bit more later.
53:29 - 53:35: So, biochemically, we validated the screening interactions, so this was done by a two-hybrid approach.
53:35 - 53:47: We verified it by co-immunoprecipitation assays, and you can see from here, FEZ1 and CRMP-1 can interact with each other in mammalian cells.
53:47 - 53:59: And we managed to map down the interaction region on FEZ1 as the core domain, which is involved in many other interactions as well.
53:59 - 54:09: We further validated this by doing reciprocal immunoprecipitation, and we were also able to show that if you pull out CRMP-1, rather than FEZ1,
54:09 - 54:13: the two proteins can also interact.
54:14 - 54:29: And very interestingly, we still don't fully understand, but the microtubule binding domain itself is important for interaction of FEZ1 and CRMP-1 itself.
54:30 - 54:48: Of course, validating the biochemical interactions, we also went on to do neurons, and very interestingly, we were able to show that FEZ1 and CRMP-1 not only localize to axons themselves, but also very importantly, they co-localize
54:49 - 55:03: in regions that respond to signaling molecules to direct growing neurites to their target areas.
55:03 - 55:09: And what's also interesting is, a while ago, and I mentioned this a little earlier,
55:09 - 55:14: we identified FEZ1 interaction with Syntaxin-1.
55:14 - 55:22: And we also showed that, you know, this interaction occurs quite prominently in growth cones as well.
55:23 - 55:34: And what's interesting about Syntaxin-1 is that a couple of years ago, the group led by Soriano also showed that Syntaxin-1 is a key factor in the Semaphorin signaling pathway.
55:34 - 55:56: As you can see in this highlighted region over here, the Syntaxin-DCC complex is required for the success of the guidance cues in order for it to respond to chemotactic and chemorepulsive pathways.
55:56 - 56:06: So this is extremely interesting that FEZ1 could be acting downstream of guidance cue pathways.
56:06 - 56:13: So just to briefly mention the importance of guidance cue pathways.
56:13 - 56:27: As I mentioned earlier in the introduction, during the development of neuronal networks, immature neurons have to start forming connections between local circuits and also global circuits if you like.
56:27 - 56:44: So the graph on the left here shows you two examples involving the cortical networks, where neurons in particular regions of the cortex itself have to either extend out to another region of the cortex itself or to another
56:44 - 56:52: region of the brain itself, and the neurites have to grow, and sometimes they will grow.
56:52 - 57:08: Sometimes they start to form branches because they need to connect to other regions, and sometimes the branches disappear or are eliminated because they are either not required or are projected to the wrong area.
57:09 - 57:26: The guidance of neurites to these regions depends on guidance cue signaling pathways, of which some are known in this regard, such as Semaphorin 3, as well as other canonical guidance cue signaling pathways.
57:26 - 57:41: And what is really interesting is that, be it Semaphorin or other cues acting through the cognate receptors downstream of the signaling, there are impacts on cytoskeletal organization itself.
57:41 - 57:59: And what is also very interesting is that Semaphorin or other signaling pathways actually contribute to both axonal growth, but it also contributes to dendrite growth, as well as synaptogenesis itself.
57:59 - 58:16: So, just showing a little bit over here, in the instance of Neitrin, there are chemotactic and chemorepulsive cues depending on the context and environment itself, which is important to bring neurites side towards the target, or again, away
58:16 - 58:22: from the targets as these form normal neural networks.
58:23 - 58:38: This led us to a hypothesis that FEZ1 could be a convergence point for signaling pathways in order to regulate transport of cargoes towards extending axons and neurites.
58:38 - 58:57: And to further strengthen this point, we were also able to see that FEZ1 itself can form complexes with the Semaphorin receptors shown over here. So here we use a co-IP method to show that Syntaxin-1 and Neuronal Receptor 1 can interact with
58:58 - 59:06: themselves. And we also showed that through Syntaxin and DCC, FEZ1 is also able to form a complex with the netrin receptor.
59:06 - 59:19: Even more importantly, SorCS1 itself, which is a common factor downstream of the Netrin and Semaphorin signaling pathways can form a complex with FEZ1.
59:19 - 59:38: To strengthen this further, we again went back to immunofluorescence, and we were able to show that FEZ1 can co-localize with SorCS1, again strengthening the idea that in the growth cones, where the guided cue pathways act on a complex
59:38 - 59:52: with downstream factors of Neurotrophin and Semaphorin signaling pathways, can be involved in delivering cargoes to support axon growth.
59:52 - 59:54: So to test this functionally,
59:54 - 60:00: we again generated the knockout neurons for FEZ1 itself. I'm not going to cover it,
60:00 - 60:07: I'm happy to discuss this later, but in terms of time, I'm going to show you the FEZ1 data.
60:07 - 60:25: So what we did was after generating the FEZ1 knockout, we added that Semaphorin 3A. So as you can see in the show clip over here, the addition of Semaphorin 3A and Netrin increases dendritic proliferation.
60:25 - 60:35: But once you take out FEZ1, you can see that the neurons no longer respond to netrin and Semaphorin stimulation.
60:35 - 60:54: This is also the case for the axons as well. When you take out FEZ1, if you treat it with Netrin and Semaphorin, the neurons no longer respond to Semaphorin-induced proliferation of axons and branching itself.
60:54 - 61:07: Now, in the case of CRMP-1, we only saw the loss of response to Semaphorin 3A stimulation but maintained the ability to respond to Netrin signaling states.
61:08 - 61:32: So, collectively, what we were able to show was that if you knock down FEZ1, the ability of growing axons and dendrites to respond to signaling cues becomes abolished.
61:32 - 61:47: Because the signaling cues themselves are important in the formation of neuronal networks. What we now hypothesize is that in patients who have FEZ1 mutations,
61:47 - 62:05: the ability to respond to guidance cue signaling can ultimately result in altered neural networks that form the basis of neurodevelopmental disorders, like the phenotypes that are seen in, for instance, Jacobson syndrome patients.
62:05 - 62:11: So I'm going to branch a little bit more for the rest of the time.
62:11 - 62:28: To look another aspect of FEZ1 involvement in neuronal network formation. And this is related to the locomotion and psychomotor impairments that have been documented in Jacobson syndrome patients.
62:28 - 62:42: So, I alluded to the idea that locomotion effects are present in model organisms that are mutants for FEZ1.
62:42 - 63:01: So, if you do a quick lookup search on gene expression in humans, using GTEx, it's actually quite interesting that FEZ1 expression in the human spinal cord is actually extremely high, likely the highest in the whole organism itself.
63:02 - 63:25: As I mentioned, the locomotion defects have been documented previously, and we wonder whether the psychomotor impairments in GS patients could be linked to the idea that FEZ1 loss could affect the development of the peripheral nervous system itself.
63:26 - 63:44: So, to first document this, we again looked at protein expression, this will come from rodents from rats themselves. And you can see that there is substantial expression of FEZ1 in the spinal cord, and you know, by immunochemistry, you can see this expression, again supporting the idea that FEZ1 is expressed in motor neurons.
63:45 - 64:06: So, we then decided, you know, to be a bit adventurous, and we started to modify a protocol to generate motor neurons from stem cells
64:07 - 64:31: And this suggests QC to tell you that we were able to get the progenitors, but more importantly, I want to show you this slide to highlight to you that FEZ1 expression does not occur in progenitor cell types, as you can see, it's not detected in your epithelial
64:32 - 64:44: motor neuron progenitor cell types, but the moment they commit to motor neuron lineage, FEZ1 expression starts to appear and it increases over the maturation of these motor neurons.
64:45 - 64:53: What is interesting is when you do an FEZ1 knockout by CRISPR, as I mentioned to you previously in the rodent neurons.
64:54 - 65:07: By western blot you don't really see much change, except for the fact that no FEZ1 expression is abrogated, indicating that the conversion to motor neurons can still happen.
65:08 - 65:23: But if you look at it phenotypically, again, we're registering developmental defects in the form of axonal developmental delays, shorter neurons, shorter axons, shorter dendrites.
65:24 - 65:38: And if you look at the dendritic development itself on the right, you can see that the FEZ1 knockout neurons dendritically develop a lot slower as compared to the wild type or the control neurons themselves.
65:39 - 66:00: And I mentioned previously that FEZ1 is important for transport. So we started to look at this again in motor neurons, and you can see that in wild type motor neurons, the punctate distribution of FEZ1 in the wild type motor neurons along axons and dendrites
66:01 - 66:17: themselves. And if you look at co-localization of this puncta with their cargos, one of which is Piccolo, which is actually some protein, we can see that there’s quite a lot of co-localization between Piccolo and FEZ1 itself, which again supports the idea that
66:17 - 66:23: in motor neurons, FEZ1 is involved in the transport of synaptic cargos.
66:23 - 66:45: But it’s also interesting that if you look at the transport and we did this by correlating the extent of signaling of fluorescent signals in FEZ1 knockout neurons with control neurons themselves, you can see that the appearance of cargos in both
66:46 - 67:10: proximal and distal regions of the knockout neurons are significantly delayed. So, whereas Piccolo cargos can be already detected at day 9 in control neurons, the signal for Piccolo cargos in the proximal axons only appears at day 21.
67:10 - 67:26: And as you can see from the quantification charts over here, the delay is substantial, both in proximal and also, of course in distal axons themselves, indicating a disrupted synaptic transport.
67:26 - 67:45: So, I'm going to skip this, just to mention that, while previous papers have shown that FEZ1 mutants in *C. elegans* and Drosophila have been documented with locomotor defects, they have never been systematically characterized.
67:45 - 68:01: So what we wanted to do was to really look at this data again. And one of the first things that we did was to use the germline FEZ1 mutant. And again, if you do the germline mutant, which is the strongest, you can see that there are crawling defects in the
68:01 - 68:19: larvae, again, supporting previous publications, indicating that when FEZ1 is gone from organisms, there are locomotion defects. But what was also interesting is that the survivability of these organisms actually dropped drastically.
68:19 - 68:29: So, we wanted to move away from whole body knockout and decided to do motor neuron-specific knockdowns, so these are siRNA constructs.
68:29 - 68:49: And you can see that when FEZ1 expression is specifically reduced in motor neurons in the fly, there is still sufficient to cause locomotion defects either in terms of crawling or climbing in the flies themselves, and again, the viability
68:49 - 69:09: also dramatically drops as has been observed in the germline knockouts. What is even more interesting confirming the observations that we have seen in the human motor neurons is that neuromuscular junctions, the formation of muscular junctions, are impaired.
69:09 - 69:16: So you can see that this is the wild type, and this is the siRNA for FEZ1.
69:16 - 69:24: And you can see that the number of synaptic boutons at the neuromuscular junctions drops dramatically.
69:24 - 69:41: So from our previous work, we found that FEZ1 phosphorylation is required for its function. And one of these kinases is ATG1.
69:41 - 69:46: And ATG1 is amenable to activation by pharmacological intervention.
69:46 - 69:56: So what we decided to do was to treat these flies with ATG1 activators.
69:56 - 69:59: And as you can see over here.
69:59 - 70:19: So, if you treat the flies with either Rapamycin or Metformin, the viability is not significantly affected, but you can see that locomotion, in terms of climbing itself, improves significantly, and also more importantly, the bouton numbers
70:19 - 70:25: at the neuromuscular junctions appear to improve as well.
70:26 - 70:48: Taken together, these findings actually demonstrate two things. One is the possibility that the locomotor and psychomotor impairments in GS patients can be attributed to the effect of loss of FEZ1 in motor neuron development, as well as
70:48 - 71:01: the activation of neuromuscular junctions, and the possibility of potentially intervening with pharmacological treatment in the patients themselves.
71:01 - 71:04: So I'm going to leave you with the summary slide.
71:05 - 71:25: I hope I managed to show two things. One is the definite role and importance of FEZ1 in the development of neuronal networks itself in the CNS. We show in addition to the role of FEZ1 in transporting synaptic proteins and in the formation of synapses.
71:26 - 71:46: The idea that FEZ1 is the hub or central protein that responds to guidance cue signaling, thereby allowing the formation of normal neuronal networks, and in the PNS building upon this again to show that this is not just important for the formation
71:47 - 72:03: but also that the defects in FEZ1 mutants could be related to psychomotor defects that can be found in human patients themselves.
72:03 - 72:10: So, last but not least, I'd like to acknowledge the people who have done the work.
72:11 - 72:16: The work on guidance cue signaling was performed by Chua Jie Yin, a former student RA of the lab, and Ng Shi Jun, who's currently still with us as a final year project student with a contribution from Oleksandr Yagensky, a previous PhD student.
72:30 - 72:59: The work on the motor neuron project was helmed by Sara, who's a very talented PhD student, now an RA in my lab, with Rafhanah, who's a PhD student, and supported by Wang Ziyin and Venetia, and Sylvester and Sumitra, who work with us.
73:00 - 73:13: And of course, I'd like to acknowledge the funding from the various organizations that have supported our work, and to all of you for your kind attention, and thank you.
73:13 - 73:18: Looking forward to the questions, if there are any.
73:18 - 73:26: Thank you Jennifer, your presentation was really interesting to hear about the different roles of FEZ1 in neuronal circuit formation.
73:26 - 73:33: We already have a few questions coming through, so I will jump right in and ask those questions.
73:33 - 73:44: A very simple question that I will ask first is, whether FEZ1 knockout mice, whether they are embryonic lethal?
73:44 - 73:59: They are not embryonic lethal; actually, they can be born. And what is really interesting for us is that we didn't, I mean, this was published by another group some years ago.
73:59 - 74:21: And what was really interesting was that the defects in the FEZ1 mutant mice appear to be actually surprisingly mild in fact, and we are considering the idea that, you know, FEZ1, because of its role as an integral hub protein, knocking it out so early
74:22 - 74:43: might actually cause compensatory mechanisms to kick in, and this has been shown in other mouse models as well. And we're actually wondering whether the low effects are being masked by these compensatory mechanisms.
74:43 - 74:45: That is a very good.
74:45 - 74:56: Another question that came through is about the protein-protein interactions FEZ1 has, and whether there's an interaction with synaptophysin.
74:57 - 75:16: This question is a bit longer, so I will read it out. One person from the audience said, “Our molecule NRP2 and Semaphorin 2945 provoke shifting of somatically localized synaptophysin to the periphery in cultured adolescent CNS neurons.
75:16 - 75:21: I wonder whether FEZ1 is crucially involved in mature synaptogenesis.”
75:21 - 75:37: That's a good question actually. You know, when we first... So, okay, first question, because we don't have direct evidence that FEZ1 is interacting with synaptophysin.
75:37 - 75:41: So I can answer that.
75:41 - 75:47: But whether or not it's involved in mature synaptogenesis—
75:48 - 75:58: There is some indication that it could be partly from us and probably from the group
75:59 - 76:13: they have done some studies on this one and FEZ1 a number of years ago, a very interesting study suggesting that, you know, FEZ1 is involved in adult neurogenesis.
76:13 - 76:23: And this was really very interesting for us. And when we first saw this, and we looked at our data in early neurogenesis, if you like, early development, there's a bit of a contrast, which probably indicates that, you know, FEZ1 has two
76:33 - 76:52: roles, one in early neuronal development and one in late neuronal development, as in adult neurogenesis. So I think there is some involvement; by exactly how that happens, I think we're at the moment still not sure.
76:52 - 76:59: So, maybe also in connection with this. Is there anything known about the regulation of FEZ1 function?
76:59 - 77:19: Yeah, so I alluded to this briefly just now that we and others have shown that there are at least two kinases involved in FEZ1 regulation. So the ATG1 finding was found by Toth et al.
77:19 - 77:32: And we actually make use of this finding in order to show that, you know, a pharmacological intervention of ATG1 modulators can rescue locomotion defects.
77:32 - 77:44: And then we also found another kinase, the MARK kinases, the microtubule affinity regulating kinases, which I think some of the audience know are TOLL kinases.
77:44 - 77:58: And this is actually one of the findings that led us to associate FEZ1 dysfunction in our axonal transport defects and ultimately to Alzheimer's disease in the AD transgenic mice.
77:59 - 78:05: There is another form of regulation that has been reported. And this is the ubiquitin relation.
78:05 - 78:12: And it's been shown that regulatory, rather than the so-called degradation of FEZ1 itself, is required for FEZ1, meaning—sorry, I just correct myself—meaning that the ubiquitin relation of FEZ1 is required for neuritogenesis as well.
78:34 - 78:35: Okay, thank you.
78:35 - 78:48: Another question from the audience is around the contexts of FEZ1 transporting synaptic cargo, and how they contribute to axonal phenotypes, including neurite extension.
78:48 - 78:59: And it’s a very good question. So, I briefly mentioned that there are synaptic cargoes that we picked up before.
78:59 - 79:13: And I also mentioned that if you look at the by ontology, at least quite a number of these proteins also play important roles in neuronal development.
79:13 - 79:26: So it's quite well known that, you know, synaptic proteins don't just play a role in synapse formation or some function, but they do have a role in, you know, in axonal and dendrite formation.
79:26 - 79:37: So, I think there are two possibilities here. One related to the idea that FEZ1 is required to bring in the raw building blocks
79:38 - 79:56: for axonal and dendritic development to occur. So, a purely transport role itself; but once the cargos actually pass on to the sites of growth, they also help to promote on axonal dendritic development, which
79:56 - 79:59: is required for the actual synaptogenesis to form.
80:00 - 80:14: Okay. And one last question I have is around the diseases of FEZ1's involvement. We talked a lot about the neurodevelopmental disorders, and you also brought up Alzheimer's disease.
80:14 - 80:20: And I wonder whether FEZ1 would maybe also then play a role in other neurodegenerative diseases.
80:20 - 80:26: Yeah, so it's also a very interesting question because, what we're actually seeing.
80:26 - 80:43: I mean, there have been reports of FEZ1 malfunction in, for instance, ALS. FEZ1, I didn't mention is also has been reported by Ghosh and his group as a regulatory compound of Kindlin-1 function.
80:44 - 80:58: So it would appear to be, especially taking the context of the idea of our AD findings that if you affect the regulation of FEZ1 itself,
80:58 - 81:03: that in turn can affect Kindlin-1 function.
81:04 - 81:12: The suspicion is that because axonal transport defects are quite commonly identified in neurodegenerative disorders,
81:12 - 81:17: we might see more FEZ1 involvement in other degenerative disorders as well.
81:17 - 81:22: Okay.
81:22 - 81:26: Thank you. Yeah, that's very interesting insights.
81:26 - 81:29: Again, thank you for your fascinating talk.
81:29 - 81:33: With this, I will move to our last speaker for today.
81:33 - 81:47: Our last speaker for today is Dr. Noriko Osumi, and she is Vice President at Tokyo University in Japan, and a professor for Development and Neuroscience belonging to Tokyo University's School of Medicine.
81:48 - 82:00: Her research background is developmental biology, and she has an interest in brain development, evolution, and disease. Manipulating mammalian embryos is one of the key expertise in her lab.
82:00 - 82:10: And I now look forward to Noriko's talk on the effect of paternal aging on transgenerational epigenetics.
82:10 - 82:14: Thank you, and Noriko, and I'm very happy to see you online.
82:15 - 82:21: So, I'd like to share my slides first.
82:21 - 82:24: Can you see my slide?
82:24 - 82:25: Yes.
82:25 - 82:33: Okay, thank you very much. So, I'm so much honored to give a talk online today, supported by Abcam.
82:34 - 82:40: Actually, so, we have used the outcome antibodies so much.
82:40 - 82:51: And so actually, this is my good opportunity to introduce our recent work to the audience.
82:52 - 83:04: And so, as the previous two speakers were more focused on the genetic background for the risk factors, some neurodevelopmental disorders,
83:04 - 83:12: but today I'm going to talk about more epigenetic components. Okay.
83:12 - 83:18: So, as you know, mental health is a very important issue in the world.
83:18 - 83:23: There are so many people who are suffering from various mental diseases.
83:23 - 83:38: Of course, depression is most of the largest number, but in the case of the neurodevelopmental disorders, I think that this is a very important issue, which has a very important social impact.
83:38 - 83:53: And also, some of these mental illnesses are originated from the impairment in the developmental period, as previous speakers have already taught.
83:54 - 84:13: Therefore, I would like to emphasize again that neurodevelopmental disorders are so complex, with various names of disorders and also with various other symptoms as well.
84:14 - 84:27: But I just would like to emphasize the importance of autism and other related neurodevelopmental disorders because there is a pandemic rise in the number.
84:27 - 84:34: In this case, this is a number in the US, and actually more and more in recent days.
84:35 - 84:50: So, this increase in the very short period is not just due to a diagnosis, and it's too short for the change in the genetic background.
84:50 - 84:53: So, there could be something else.
84:53 - 85:02: Again, I just would like to mention that the symptoms of ASD, for example, are very complex.
85:03 - 85:18: As you know, ASD is diagnosed as a kind of social impairment, including some vocal communication as well, and also some other repetitiveness, something.
85:20 - 85:30: For example, their playing style is so much limited in diversity.
85:31 - 85:52: But actually, there are other symptoms like the sensitiveness, or some awareness, or it's not shown here, but some ASD kids also suffer from sleep defects and digestive defects as well.
85:52 - 85:54: So, it's so complex.
85:55 - 86:10: And also, I'd like to mention one thing that the genius of genius people, as shown here, is atypical in a way.
86:10 - 86:13: So, it's a kind of a spectrum.
86:14 - 86:17: Well, who is considered a patient?
86:18 - 86:21: And, and the genius people. Okay.
86:22 - 86:33: As Noriko has already mentioned, my laboratory has so much interest in brain development, and I have been a researcher in this field for a long time.
86:34 - 86:55: My favorite gene is PAX6, which is a transcription factor, and which is important in brain development and CNS development, but it is first identified as a responsive gene for the, as a master gene for eye development.
86:56 - 87:19: I'll come back to that later. So, and PAX6 is important in various aspects of brain development. For example, the patterning of the neural tube, and neurogenesis, and it is also involved in cerebral development and astrocyte maturation as well.
87:20 - 87:37: Because PAX6 is important for eye development, mutations in PAX6 have been reported so many times in patients with the eye problem, which is called aniridia, especially.
87:38 - 87:55: So, and so this summarizes the case reports. The ocular phenotype is so obvious; that is why the patient comes from the eye doctor, and some patients are identified as having PAX6 mutations.
87:56 - 88:09: But also, there are some people who have neural phenotypes, such as mental retardation, learning defects, or aggressiveness, etc.
88:10 - 88:18: And that is why we have started to look for PAX6 mutations also in the Japanese patient population.
88:19 - 88:34: PAX6 is relatively large as a gene, consisting of 15 exons. So we have sequenced from the first exon to the last one, including the fronting introns.
88:34 - 88:50: And then we came up with 15 unique SNPs within the 285 SNPs, which were not found in 2,120 non-ASD cases.
88:50 - 89:08: So here I showed one missense family, where the daughter is diagnosed with autism, and that mutation is derived from the father, and both of them are showing the eye phenotype, but the father is not diagnosed with autism.
89:09 - 89:32: So there could be something, not just a genetic component. But that's the starting of the beginning of the autism research. We first did the behavior analysis using the PAX6 mutant we have already identified as an original one.
89:33 - 89:55: We have done many other behavior tests and found that such as these mutant rat of PAX6 show reduced interest, social impairment, vocal communication defects, sensory motor gating abnormalities, and also some memory defects.
89:55 - 90:07: It also shows a reduction of the serum serotonin levels, which I'm not discussing today. So we consider that PAX6 mutant rat are a good model for autism.
90:08 - 90:32: But there are already so many genes related to the risk of autism, as previous speakers have already mentioned. We consider that PAX6 is just one of them. So there is so much complexity of the combination of these risk factors. So what shall we do?
90:33 - 90:52: We have now focused on more non-genetic factors, and one of them is, of course, the maternal component; there's some defects, such as some drug exposures or nutrition defects that are already reported.
90:53 - 91:04: So, we have more focused on the more rare cases, how to say, cases which have not had so much attention at the moment, you know, like 10 years ago.
91:05 - 91:28: So, the paternal aging has a risk. This is the case in Japan, and the first age of marriage is increasing in males and also females. Consequently, the age of the first child is increasing.
91:29 - 91:47: On the right side, I'm showing a meta-analysis of more than 5 million people showing that the risk of offspring developing ASD is larger in fathers rather than in mothers.
91:48 - 92:06: So, if the risk of the maternal and paternal age is considered as one in the 20s, it increases according to age, but the father's risk is larger rather than the mother's.
92:07 - 92:33: Okay, so, and there's another paper that, this is also the meta-analysis of mother age reports. Actually, paternal age and smoking are connected to low birth weight or smaller gestational age, and these factors also have subsequent outcomes as well.
92:34 - 92:51: In fact, the advanced paternal age impacts various mental disorders, not only autism spectrum disorders, but also schizophrenia, bipolar disorder, etc.
92:51 - 93:14: There have already been some reports focusing on paternal aging, and there are various phenotypes that are already reported, which are rather very complex.
93:15 - 93:26: But first, we would like to add that paternal aging is actually a confounding factor in combination with the PAX6 mutation.
93:26 - 93:46: We did the analysis of behavior using the pups derived from IVF, using the sperm from the PAX6 heterozygous father and using wild-type eggs.
93:47 - 93:59: The outcome can be the 50-to-50 ratio of the offspring, either from the heterozygous or the wild-type.
93:59 - 94:15: And then we had the phenotype difference between the offspring if the father or the sperm is from the young or aged male mice.
94:15 - 94:35: In the case of young fathers, this is more common to see vocal communication, but in the case of aged fathers, offspring from the heterozygous offspring and the wild-type ones, show hyper locomotion.
94:36 - 94:43: So, paternal aging is an important confounding factor; this is an important thing.
94:44 - 95:10: So, for those people using animal models, we try to use, for example, transgenic mice, etc., so using the male mice for a very long time, but when they become very old, we need to know that the phenotype can be different if the father is aged.
95:11 - 95:27: This introduces another issue for human genetics as well, because paternal aging can, how to say, cover the actual phenotype due to the genetic background.
95:27 - 95:46: Okay, so about 10 years ago, we started the paternal aging project, and these are my team. The photo was taken before the coronavirus. I hope that we would like to come back to this situation soon.
95:47 - 96:11: Okay, so now we would like to focus on what are the factors that relate to paternal aging. To do that, we first set up using wild-type mice and using 12-month-old mice as corresponding to the late 50s to early 60s in humans.
96:11 - 96:26: And then we got the, we first analyzed behaviors. Before that, this is rather a kind of mild state of aging, okay? So it's not like 80 years old.
96:26 - 96:42: So, as a result, the litter size and body weight litter size of the offspring is not different, but the body weight is slightly lower in the aged father-derived ones.
96:42 - 97:02: In this situation, we did various behavior analyses and this is just a summary of the table. The ultrasonic vocalization is reduced, the pre-pulse inhibition is reduced, showing the sensory motor gating phenotype, and learning defects are observed.
97:03 - 97:29: However, if we analyze the F2 offspring, which means that the grandfather is aged but the father is young, they did not show the behavioral phenotypes, but only the lower body weight remains.
97:29 - 97:40: We consider that some more epigenetic mechanisms can be the reason for these kinds of behavioral phenotypes.
97:41 - 97:59: Here, I just would like to emphasize a very interesting phenotype of the USV, because these are very automatic recordings, okay? Just five minutes in this recording box.
97:59 - 98:20: We are using the maternal separation-induced pups USV because this is considered to be equivalent to the babies crying in human beings. It can be recorded at a very early stage, okay? We start from P3.
98:21 - 98:29: So, I just would like to, ah, sorry. Yes. Can you hear the sound?
98:30 - 98:43: So, we recorded this kind of sound in the experiment, it was untouched, okay?
98:44 - 99:02: What is nice here is that we can collect a lot of data from just five minutes of recording. First, we use Fourier transformation, and then we did analysis of the imaging analysis.
99:02 - 99:13: We can also use kind of a machine-learning type of image analysis, which is very powerful in identifying even the tiniest phenotypes.
99:14 - 99:30: Okay, so today's time is limited. I’ll only present a small number of the data. The number of the syllable types, which means the variation of the syllables, is reduced in the pups derived from the aged father.
99:30 - 99:45: This is another example using entropy analysis. In this case, these are kind of maps showing each of the calls of the young father-derived ones and the aged father ones.
99:45 - 100:00: Starting from P3 to P12 in a postnatal context, you see that there is convergence in the recordings of young father-derived ones, which is not so obvious in the case of aged father-derived ones.
100:00 - 100:04: This is another example:
100:04 - 100:07: In this case, one dot shows all the data compressed in two dimensions,
100:07 - 100:18: where one pup shows one dot.
100:18 - 100:27: At P3, you can see there is some variation in vocal communication in the pups.
100:27 - 100:28: But in the case of young father-derived PUPs, the variation is reduced and converges into a relatively small space.
100:28 - 100:37: This means that there is more typical development.
100:37 - 100:49: But in the case of the aged father-derived PUPs, which is shown in the red dot, at first
100:49 - 100:54: there is some overlap, almost indistinguishable from the young father-derived PUPs, but
100:54 - 101:08: gradually, there is still a significant atypical development.
101:08 - 101:16: So we consider that the father's aging has induced more atypical individuals.
101:16 - 101:24: We understand that paternal aging alters the vocal communication
101:24 - 101:27: in the infant mouse.
101:27 - 101:34: This relates to ASD-like impairments, especially in cerebral development and diversity,
101:34 - 101:39: and increased individuals with atypical vocal patterns.
101:40 - 101:47: And then, we confirmed that, okay, so 12-month-old is a good model.
101:47 - 101:52: Not too old, but it's considerably reasonable.
101:52 - 101:58: We would like to ask what factors come from the father.
101:58 - 101:59: Of course, there are lots of steps from the father to the offspring behavior.
102:07 - 102:16: So first, we focus on the epigenetics of the sperm because in 2012,
102:16 - 102:24: some reports showed that paternal mutations increase with age, but over time, there are reports showing that those de novo mutations due to advanced paternal age are not as much a risk for ASD in the offspring.
102:39 - 102:42: So we want to focus more on the epigenetics.
102:43 - 102:54: Among the epigenetic components, we identified this paper showing that paternal sperm DNA methylation is associated with early signs of autism risk.
103:01 - 103:08: These changes in DNA methylation are also observed in the post-mortem brains of ASD patients.
103:10 - 103:17: There are more epigenetic factors, but we first focused on DNA methylation.
103:18 - 103:28: We took sperm from young or aged mice, and then we did whole-genome methylation analysis using the SureSelect type of mutation.
103:36 - 103:41: I'm very sorry that this is Japanese, but okay, I will explain.
103:41 - 103:51: We came up with 16 hyper-methylated regions, but we came up with more than 96 hypo-methylated regions in sperm DNA.
103:54 - 104:04: Very interestingly, if we did a gene term analysis, we came across a lot of nervous system-related gene terms.
104:11 - 104:19: Okay, so another thing is that we have a common feature, which is an REST-binding sequence, as shown here.
104:23 - 104:31: These also show up in other analyses using a ChIP atlas in silico analysis.
104:31 - 104:38: By analyzing our hypo-DMRs using the data uploaded in the ChIP atlas, we found high significance.
104:49 - 104:55: Next, we analyzed transcriptomic data using the embryonic brain, and we did RNA-seq analysis.
105:09 - 105:14: Fortunately or unfortunately, we did not see very rare cases that had high significance and high fold change.
105:22 - 105:35: But we noticed a significant upregulation in the aged father-derived offspring with neuron-related genes.
105:43 - 105:51: We conducted gene set enrichment analysis (GSEA) and found that late-fetal genes are enriched in the aged father-derived offspring, but not in early-fetal genes.
106:02 - 106:10: Additionally, aged father-derived offspring show enrichment in ASD-related genes, which in this case, we used the Safari database.
106:13 - 106:20: These results consistently demonstrate a precocious neurogenesis in the aged father-derived offspring.
106:24 - 106:32: We found that the REST-related genes were upregulated in the aged father-derived offspring.
106:35 - 106:47: What is REST? REST is important for brain development.
107:10 - 107:16: This is from a paper published in Cell in 2012. We consider that REST is a key epigenetic mechanism by which paternal age impacts embryonic developmental programs.
107:28 - 107:44: To know whether DNA methylation is indeed responsible for the offspring's behavior, we conducted analyses using the demethylation drug treatment for the young mice.
107:53 - 108:06: So, we found that sperm became less methylated, and
108:06 - 108:14: In the right side, this is the demethylation, the marker is upregulated like this.
108:15 - 108:21: So, in this condition, I'm sorry, I like to wrap up.
108:21 - 108:27: And so, again, the litter size is not so much different, and in this case, the body weight
108:27 - 108:34: is not so different, but we came up with the, oh, sorry, the defect in the ultrasonic
108:34 - 108:37: vocalization, which I'm not talking about for a minute.
108:38 - 108:45: Okay, so and the last confirmation is actually, so demethylation is seen in the REST target
108:45 - 108:45: genes.
108:45 - 108:53: So, we have analyzed these 10 or so genes and came up with these two genes are actually
108:54 - 108:56: demethylated like this.
108:58 - 109:00: So, this is a control one.
109:00 - 109:05: Okay, so I'd like to skip this, and actually, so these are the REST genes.
109:05 - 109:13: Okay, so this is a summary of today's talk, and so I think that this is a possible scenario
109:13 - 109:23: that in the case of aged fathers, there are hypermethylated regions more, and the common motif is a REST target
109:24 - 109:31: target motif, and this influences brain development through the REST target genes.
109:32 - 109:44: So, and so this is published, it's also presented from the EMBO, and also, I just finally would
109:44 - 109:52: like to mention that, so this paternal aging is the origin of the neurodiversity, because
109:52 - 110:00: during brain evolution or over a longer scale of human history, there is
110:00 - 110:07: more neurodiversity coming from the longer longevity of people.
110:08 - 110:17: So, I just, I don't like to have to say paternal aging is just a risk, but actually, paternal
110:17 - 110:27: aging has a very significant impact on human evolution as well. So, I also had published
110:27 - 110:33: this commentary paper, so if you have interest, please refer to it. Okay, so thank you very much,
110:33 - 110:40: and I'd like to thank my collaborators and the funding, and also the lab members. Thank you very
110:40 - 110:53: much indeed. Thank you, Noriko, for your very interesting presentation. We have some questions.
110:54 - 111:02: The first question is related to the increasing number of autistic people, and the question is,
111:02 - 111:09: is it related to better detection? Yes, that's true, but because the determination of the, how to say, diagnosis has changed since 1980, and then,
111:19 - 111:25: but still we are seeing an increase year by year, so we consider that there could be something
111:25 - 111:34: else as well. That is my opinion. Yeah. Do you have, yeah, any explanation or idea what
111:34 - 111:41: these other factors could be? Well, I think that there could be some, like nutrition,
111:41 - 111:49: it’s also some effect, and also the, how to say, use of some mental disease drugs
111:49 - 111:58: for pregnant mothers can also have an effect. Yeah. Yeah, and I wonder why does the
111:58 - 112:04: paternal age have a more significant impact than the mother's age on the prevalence of ASD?
112:06 - 112:12: Well, that's an interesting question, and in the case of the mother's age, that matters more
112:12 - 112:22: severely, which means that it's not inducing very mild phenotype-like mental diseases.
112:22 - 112:29: It's rather having more severe defects. For example, the typical example is Down syndrome,
112:29 - 112:37: right? So, the more severe phenotype can be seen in the case of the mother's age,
112:37 - 112:44: and also if the mother's age, and then the conceptus cannot survive during pregnancy.
112:45 - 112:52: So, that way, I think that paternal age has a more, how to say, the impact on making diversity, of course, not only neurodiversity, but also in other
112:59 - 113:06: physical conditions as well, I think. Thank you. Another question that came through
113:06 - 113:12: is around epigenetic changes, and the question is, could a similar set of epigenetic changes be
113:12 - 113:16: found in other age-related effects on offspring? For example, Down syndrome.
113:16 - 113:25: Right. So, I just showed you just one summary slide that paternal aging not only influences
113:25 - 113:31: the risk, the increase of the risk of ASD, but also of schizophrenia, bipolar,
113:31 - 113:38: others as well, and also it induces lower body weight, and the outcome of the lower body weight
113:38 - 113:45: at birth is wider in metabolic syndromes as well.
113:47 - 113:52: And how do you think Pax6 and the lower body weight are linked?
113:54 - 114:03: Well, in the case of our previous study, just checking that young father derived paternal aging,
114:03 - 114:10: no, no, no, but in both cases, paternal, how to say, Pax6 mutation itself has not much impact on body weight. Okay. And another question,
114:18 - 114:22: what kind of epigenetic factors can be transmitted to the next generation?
114:23 - 114:30: Okay. So, today I just would like to, I just introduced you to DNA methylation,
114:30 - 114:36: but there are other epigenetic changes as well. We have already published one small paper,
114:38 - 114:45: it's kind of a catalog showing that various histone modification molecules
114:45 - 114:50: have also gone up and down in the case of paternal aging. So, we have checked that
114:52 - 114:59: just the histological analysis using testis sections, but it's a very, how to say,
114:59 - 115:04: dynamic change also occurring in histone modification as well.
115:05 - 115:09: And we are now trying to identify microRNA as well.
115:10 - 115:17: Okay. Thank you. If there are no further questions, I would like to thank Noriko for
115:17 - 115:19: her very interesting presentation. Thank you so much.
115:19 - 115:20: Thank you very much.
115:20 - 115:28: Thank you. And with this, I would like to say that it's been a very engaging session
115:28 - 115:34: with the presentations we had from our experts today. In today's session, we learned more
115:34 - 115:39: about how impaired neurodevelopmental processes can contribute to the onset of various
115:39 - 115:45: neurodevelopmental disorders. For example, we have learned how Cy5P1 plays an important role
115:45 - 115:51: in shaping social behavior, but we also heard how FS1 regulates axonal transport and neuronal
115:51 - 115:58: network formation. And just now from Noriko, we had the chance to learn how PAX6 mutations,
115:58 - 116:03: and especially epigenetics, affect the onset of neurodevelopmental disorders.
116:04 - 116:09: But at the same time, I think we can all agree that there are still many things we do not know
116:09 - 116:14: about mental health and neurodevelopmental disorders. So, I hope that future research
116:14 - 116:20: in this field will bring further advances. I would now like to take a moment and thank
116:20 - 116:25: today's speakers, Ki-Jun, John, and Noriko, for sharing their fascinating work with us.
116:26 - 116:30: And without you, today's session would not have been possible. So, we do really appreciate your
116:30 - 116:37: contribution. On behalf of Abcam, I would like to thank everyone for attending today's event.
116:38 - 116:42: We really do appreciate your feedback. So, if you have any, please let us know.
116:43 - 116:48: We will be conducting a short poll that should appear on your screen shortly.
116:49 - 116:54: We are also going to be putting the information in the chat box, should you wish to request a
116:54 - 117:00: PACE credit for today's event. As part of our Neuromonth, we have more sessions coming up.
117:00 - 117:05: And we do look forward to seeing you at the next sessions. The next session is about
117:05 - 117:13: intracellular trafficking and will take place on October 26th. There is separate registration
117:13 - 117:18: required for the next session. The link has been placed in the chat for you to register,
117:18 - 117:31: if you haven't done so already. So, again, thank you and see you then.

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