SYNGAP1 Epilepsy Research: Insights into mechanisms and therapies
Here are the introductory comments:
In this video Dr Shilpa Kadam presents SYNGAP1 Epilepsy Research: Insights into Mechanisms and Therapies. Originally shared on July 2nd, 2020. Please find additional Syngap research webinars here: https://syngapresearchfund.org/webinars
Dr Shilpa Kadam is an Associate Professor of Neurology with joint appointments at The Kennedy Krieger Institute and Johns Hopkins University School of Medicine.
Dr Kadam’s lab is focused on investigating mechanisms underlying epileptogenesis in acquired and genetic developmental disorders like SYNGAP1. The lab is interested in discovering novel drug targets and delivery systems directed at the central nervous system.
Dr. Kadam has broad clinical exposure to epilepsy management.
Her work with SYNGAP has been focused on the relationship of poor-quality sleep and epilepsy. Her laboratory was able to identify abnormal underlying brain activity and high incidence of seizure during sleep by examining mouse models and overnight EEG from SynGAP patients. Her laboratory then discovered that using a low dose of Perampanel (Fycompa) may not prevent seizures but can help correct the high-frequency abnormal brain activities during sleep in mice model. This itself may have beneficial effects during sleep of SYNGAP patients. She hopes this approach will lead to a human trial.
Dr. Kadam is also interested in antisense oligonucleotide therapy ASO to help rescue SynGAP levels in the brain.
She is currently seeking funding for several projects in SYNGAP with one of the ones more important being on CRISPR/Cas technologies to create mice models to find potential biomarkers.
Dr Kadam: My training has been in epilepsy research and it was Dr. Huganir who introduced us to SYNGAP1 research and the mouse that the data we will I'll be showing today and we will be discussing is the is the mouse that his lab generated quite a few years ago but it has a very critical mutation in a very critical domain that allows us to study almost the most critical functions for SYNGAP1 which has been very well documented by all his early research and continuing research. So what I'm hoping to try and communicate today is how we approach this research. Why did we do it the way we did it and some of the insights that are maybe very novel that might generate new hypotheses that my lab, myself will pursue in the goal of therapies. And as parents you guys know that, you know, understanding how this protein is affecting the brain development is very critical but at the same time if we can try and figure out what new therapies might help improve, even by a little bit, the incidence of seizures in children with SYNGAP1 mutations is also important.So my slides are not moving forward... okay. So all of this information is known to you but I'm going to give you a background just so that you understand what we are focusing on. So just like Sydney said seizures and sleep disorders are predominantly one of the spectrum of SYNGAP1 haploinsufficiency and myoclonic and absence seizures are the dominant phenotype both described in Ingrid Scheffer's first cohort study and I see it as the description for all the reports that I get for the EEGs that the parents have sent us copies for so far. And of course in epilepsy research there's a long-standing relationship between epilepsy and sleep problems especially in neuro development disorders.
So at Kennedy Krieger we have huge focus on Rett Syndrome and and there are many similarities in the sense of the progression of the epilepsy, the progression of the learning disabilities, and some of the sleep disorders that have been reported by parents and have been phenotype by the clinicians who are looking at cohorts of patients with SYNGAP1 mutations. Sleep disturbance. I don't have to report it. In 100% of the patients problems are associated with sleep initiation and frequent night awakenings. Early morning seizures has also been reported and so this will become important as we try and describe some of the results we found from the analysis of overnight EEGs that parents have sent us and what we then found in the mouse model. Because for very obvious reasons once we have this phenotype there are certain investigations we can only do in the mice. We cannot do in the children. So that's where translational research becomes so important. So just as a quick background I know many of you have taken your child for clinical EEGs and overnight EEGs so you may have seen some of the trace recordings either given to you in the report or you may have actually seen with the EEG recording and I just wanted to give you a little bit of a background and hopefully you can see my mouse.
So basically right now we are focusing on wake versus sleep EEG and so wakefulness, the circuits that in our brain that promote wakefulness, usually have these very low voltage, fast oscillation kind of EEG phenotype which is right here. Wakefulness you see there is very low amplitude, high frequency discharges on the EEG. And then there are the three stages of sleep in humans, which is stage one, two and three and you can easily see that as the non-REM sleep, which is non-Rapid Eye Movement sleep, it becomes more dense, you start moving from this low amplitude, high frequency in stage one to this very high amplitude, low frequency type of sleep brainwaves. So these are very characteristic of non-REMs and then we have REM which is Rapid Eye and so the brain alternates between non-REM and REM during our sleep cycles in humans and REM you can see is almost similar to wake. So it's called paradoxical wake, paradoxical sleep because the waveform if you just looked at it and you don't know that the child at that point is awake or asleep on video you might confuse it to being wakefulness but the significant distinction is that a REM will always be between two non-REMs. So when an non-REM ends or REM starts and then a non-REM again progresses from stage 1 to 3 of non-REM and because these are associated with frequency oscillations on how the brain is firing in certain frequencies, I put in this slide on the side in blue which is all a one-second discharge and you can see what we call as "high frequency oscillation" which is gamma, which would be the focus of most of the cortical function we are trying to evaluate, both in the mouse and in the human EEGs. And so as you can see, this is high frequency. It's around 30 to 100 Hertz. Then there are other oscillations very well defined in the clinic and we try and apply the same rules to the mouse EEG. And so as you go lower in frequency it's the beta, alpha, theta but more importantly, Delta, which is the most predominant, just zero to four Hertz, is the most predominant waveform seen in non-REM stage four. So deep sleep is associated with a high density of delta. High brain activity associated with doing some tasks, being cognitively aroused is associated with high gamma.
So for this talk we are going to focus on gamma versus delta because they are the predominant frequencies in wake versus sleep. Okay. So then we'll just start straightaway with what we found with one of the overnight EEG's from this is a three-year-old child with SYNGAP1 mutation and I'm going to focus here on the middle panel where we have done the power analysis for the frequency, we had the video also, we had the notes from the clinic. And so if you just focus on so here on the x-axis this is a 20 hour long EEG, including the night. So let's say it starts late in the morning and goes on to early in the morning the next day. Okay. So that's the x axis. On the y axis are the different stages that have been scored for this EEG. So here's wake. Here's non-REM. Here's REM. So this line over here as this continues is when the child is awake. It jumps up to non-REM when the child first falls asleep and goes into the first non-REM cycle. Then as we go forward over the entire time, which is quite a few hours that this child is asleep, you can see that the child's brain is switching between non-REM, REM, back to non-REM and it keeps doing that between multiple cycles until the child is awake in the morning and that's the wake state again. So basically this entire jump here is the time based on the EEG and the video and announced the child was asleep. So then overriding that all the raster plot of the red lines shows when this child had episodes of short duration 3 Hertz seizures. Here is one example from... let's see, from this one event of this high amplitude 3 Hertz spike wave discharge on every channel. So this is a global discharge and from the background EEG which is noisy which would always be noisy when the child is awake because they're not going to stay still but what you can see in the background is this very low amplitude/ high frequency discharge which is indication that this child is awake. The cortex is in mostly in gamma frequency but in the middle of this gamma frequency this child has 3 Hertz high amplitude, short duration, 2 or 3 seconds. So that's an example of that event happening when the child is awake. Then here there's an expansion of a similar event occurring during sleep. Just on the background you can make out wake versus sleep because you have these high amplitude delta waves in the background but the seizure episode is exactly identical. It's 3 Hertz high amplitude, short duration.
So that is the predominant phenotype of seizures on this EEG and when we identify every episode that happened in the 24 hours you get these that there's an episode and as the child is coming closer to the time, even so at this point the technician in the room, the parent is does not know when the child is going to fall asleep but the brain knows when the child is, when the brain is moving towards sleep. And then you can see that there's an increase in the frequency of these discharges and then there's a short burst right when this child's brain transitions from wake to non-REM and that's here. So these are multiple episodes. That's why that line looks thick. Then what happens as we go through and as we scored them independently and just superimpose them, right when this child's brain, because now the child is asleep so there is no outside influence that would trigger a seizure, but inside brainwave states the child's brain is in non-REM, it switched to REM, that's when the brain throws a seizure. Then the brain is in REM and is switching to non-REM exactly at that transition point the brain throws a seizure. Again, another transition point there's a burst of seizures. And then again at this transition point there's a burst of seizures. And then when the brain knows it's going, the cortical state is moving from sleep to awake even before the child actually wakes up, the brain senses the strongest transition state and throws the highest bunch of seizures in this EEG.
So the point I'm trying to make is: most of these seizures are ramping up when the brain is transitioning either from wake to sleep or from sleep to wake and, very interestingly, even between sleep stages where there would not be any outside influence of light or movement or TV flash or some music. When it's transitioning from non-rem to REM and REM to non-REM this brain throws this 3 Hertz discharge. And here I have this lowest panel is just an expansion of this, what we're going to call a cluster, that now as the brain, as the cortex realizes that this brain is going to go into a wake state, it throws a multitude of these shorter shots they look just looks because it's a tighter timescale but I'm just trying to show you where four seizures within this cluster (they are not even all the seizures happening that cluster) start happening repeatedly just before the child is awake. So these might happen just when the child is going to wake up or it might happen as soon as the child is awake which is I think what the previous reports from the clinicians are where there is reporting from the parents that there is early morning seizures, difficulty falling asleep.
Now as the brain is transitioning, if the brain is seizing, there's lots of literature to figure out why then that child would not transition easily and might have difficulties falling asleep. But these are hypotheses at this point and this kind of interesting distribution is what we are trying to figure out. If we can understand better and then when we test novel therapies we can, in doing the same analysis, figure out how that is changing or not changing. Okay. So obviously the translational version of it we use a mouse model of SYNGAP1 and this mouse model like I said was generated in Rick Huganir's lab. What we are doing and what we are good at is doing these 24-hour continuous video EEGs with your EMG and so we have a 12 hour light and dark cycle, so making both the daytime and the nighttime. The technical portion is how we - this EEG signal - how we convert it here with FFT so that we can analyze all the frequencies within it. And then of course what was the title of a recently published paper is this FDA-approved drug that we tested, perampanel, which is the AMPA receptor antagonist. And we chose this drug because of all the literature that Rick's lab has already published showing that SYNGAP1 at the postsynaptic membrane, having this huge impact of how many AMPA receptors are inserted. Now AMPA receptors are, they receive the excitatory signal, so if there are too little or too many very simplistically it would completely affect how that circuit is functioning. So just not even as a therapy, we wanted to test. This is the only drug available that has this specific mechanism. And since it was approved for epilepsy very recently we thought this was the best drug, given the data from Rick's lab, to test in this mouse to see if we blocked AMPA receptors what effect it would have on the EEG and especially on the seizures.
So first what I'm showing you is video of one such seizure that we recorded in a SYNGAP1 mouse and hopefully I'll be able to play it again but what this is is the early EEG was during sleep. The mouse threw a seizure and then it transitions to - it has some spikes - but it transitions to awake EEG. So I just showed you the clinical EEG and how we scored it and about how these seizures were happening during transitions between sleep and wake and wake and sleep and so here's an example of a phenotype. Maybe I can play it again. So you can see where the seizure happens and these are very equidistant spikes associated with spikes on the EMG showing that it's an myoclonic seizure. It's a short duration seizure and the frequencies very surprisingly was 3 Hertz. So this is a 3 Hertz seizure happening at a juncture where the mouse brain is transitioning from sleep to wake.
So now the "chicken and egg question" is: did this mouse brain throw seizure and that's why it woke up? Or did the mouse brain throw seizure because the brain was transitioning and whatever is the mechanism by which the cortex changes its sleep versus wake state that makes that brain susceptible to throw seizures? So those are the questions that came to our mind when we saw very consistently the Syngap mice throwing seizures at these transition points. And then we were excited with the phenotype because it's it's a 3 Hertz seizure, it's a myoclonic seizure, and that is the predominant phenotype in the patients. So here is the same seizure without the movement so you can focus on the leads. So the first lead is the EEG lead. What I'm trying to show you are the spikes that you saw when the mouse was seizing. On the bottom is the EMG and this is to show you that when the cortex is spiking, throwing a spike, the muscles on the back of this mouse, on the neck and shoulders of this mouse, is throwing a spike (with a slight delay). So that would be the transmission of the cortical neurons firing and the muscles in the back contracting. So this had a nice correlation and the time scale here shows that this is a 3 Hertz seizure. On the bottom is just another example of a seizure where there are these random spike discharges but you can see the EMG is completely silent.
So what does this mean? That there are seizures that you will identify in your child because there will be a motor component either the eyelid myoclonia that Ingrid Sheffer has described in their paper. So there's a motor component and if you are near your child and you're observing you will know that something is wrong and that there's a seizure episode. What this data on the bottom is trying to show you is that there can be seizure activity in the cortex of the mouse for sure and the EMG silent. Meaning there is no motor component and so these could be what we call "electrographic only" seizures so they would be silent seizures. So there may be a component where there is seizure activity where there will be no behavioral component and those are silent seizures which is troublesome because if there's a huge number of these the seizure burden on the cortex makes that cortex malfunction even more because severity of seizures can affect how the cortex or the brain functions.
So this is the quantification of the seizures we found in the entire study and so it's basically because we were doing it in two-month-old mice, which is the P60 and then the same mice when they were older (P120) and you can see that over time when we recorded the number of seizure events went up and the circle here denotes the myoclonic seizures. So the younger mice had the specific just single phenotype but as they got older they started showing multiple different phenotypes. Okay. So this is what we would call progression in the mouse model. And this pie chart here is showing you that most of the seizures were starting in non-REM. And this is very very well known in both in epilepsy patients and in development disorders associated with seizures that there's something about the non-REM state with the slow oscillation Delta wave that makes the brain most susceptible to throw a seizure. So that was the phenotype in the young mouse where most of the seizures were arising from sleep and then as the mouse got older you can see that that frequency changed. Most of them were then happening during the wake state. So all this is saying is for the model that the phenotype is changing over time. And we know this to be true in developmental disorders that the epilepsy is not static. It sometimes progresses, sometimes it regresses and then sometimes the type of seizures change. So this is interesting for the mouse model because then now we are having multiple seizure types and if we do interventions we can see the effect of multiple interventions on those various phenotypes. So this is all good for the modeling world.
Mike: Dr. Kadam, I'm sorry to interrupt you but if we could just go back to slide eight for a second. For those of us who don't spend a lot of time thinking about mouse models would you mind just elaborating on what P60 and P120 mean and how those how those ages translate to kid ages because this is clearly...
Dr Kadam: That's a very important question. And I was definitely going to touch on that because Brennan is on this call and he's working on, and we are working on, doing much younger ages because of course this is a developmental disorder. So this was a pilot study. A P60 means 60 days old. So that's a two month old mouse. So that's a young mouse. So mice are weaned from their moms or "dams" at around P21 right. So that would be the time when when they can fend for themselves. So P60 is a young mouse. It is definitely not a child though. And P120 is just a older mouse and here the significance is because then we're trying to record from the same mouse to look at progression. So as a pilot study and coming from the epilepsy research field there is a very important caveat where even if you look at other papers where people have studied Rett syndrome, CDKL5, you will see that they're using adult mice. And more and more as we have these grants for review and reviewing grants, NIH and the reviewers are like "these disorders are developmental so you need to start looking at much younger ages". Luckily that is the bread and butter of our lab. We study neonatal seizures in pups but because this was our first initial foray into SYNGAP1 and nobody had quantified how the epilepsy... what was all the phenotypes possible in this model and how it was progressing over age. We needed this infrastructure to start then going uniquely. Because neonatal EEG is tricky and I will talk about that as because those are the experiments we are doing right now. So hopefully that has addressed the question but I will touch on this again when we start talking about what we are doing currently.
So that the ages we are doing right now will be covering the age even from a newborn to the first two years of life where we will want to figure out what is happening in the same mouse at early ages and can we do interventions that would then change the trajectory? But now we know what the trajectory is. So if we did some intervention in the pups and then we did a P60 again and lo and behold they had no seizures, then we we could convince the NIH or pharma that look, our intervention is changing the progression of this disease. At least for the epilepsy. And then when we talk about the gamma oscillations which totally underlie cognition that we are actually trying, we are improving cognition in the mouse.
So this data is critically important because in translational research we need this end point to show data that would have NIH's confidence or a small big pharma's confidence that we are actually by some intervention, novel or, like perampanel, that's already approved, we are able to change the trajectory of the disease. Because developmental disorders have that, that in an immature brain having seizures there's lots of literature to show the seizures themselves affect how that brain matures. And so it's difficult to parse out how much are the seizures by themselves contributing to the cognitive dysfunction versus the underlying genetic cause that may be contributing to cognitive dysfunction.
So "part and parcel" of seizures are interictal spikes. Interictal means between two seizures. So any kind of spike activity that is now identified by these red asterisks here is called interictal because now we know that this mouse seizes and so interictal spikes by themselves are a huge area of focus and research for being able to predict when a seizure is going to happen. So usually normal EEGs will not have these spikes. Or at least not at this frequency. So what this very simplistically indicates is that this brain is hyper excitable. There are times when the person or the mouse is just normally functioning that a bunch of neurons just throw a massive spike together. So this does not happen in a normal brain. This is just an example of spikes happening during wake and spikes happening during non-REM which is the sleep state we just discussed. Here again is the background EEG showing you wake. It's small amplitude higher frequency. Here's the background showing you high amplitude, slower frequency. The spikes almost look identical but what this example shows you that in general we found there were more spikes during non-REM then during make states. This again ties and is very common in epileptic brains where seizures and spikes happen during sleep states and that that circuit state is more conducive for seizures to appear.
So here Brennan went and quantified every spike in the 24-hour EEG. So this is a lot of work and it shows: so the wild-type is the mouse that does not have the mutation and then the P60 and P120 like we discussed is the mutant mouse at 2 months old and then the same mouse older. And you can see a natural progression in the incidence of these spikes but you can see it as the color code is wake versus sleep that most of these spikes and significantly are occurring during sleep. Then we also know that, you know, the light phase has an effect on spike and sleep generation. So because mice have diurnal cycle in addition to the circadian cycle, when they sleep during when the lights are on versus when the lights are off. Why is this important? Because unlike us rodents are nocturnal. So basically they're sleeping during the light and they're exploring, eating, grooming during the dark. And as you can see, most of these spikes when we parse the same data into whether it was during the light sleep cycle or the dark sleep cycle, most of them are occurring during light sleep. So this is when the rodents are sleeping. So this would mean like a child during the night if we were trying to make a human comparison. And then as we have already seen before when you look at the sleep stage within light sleep or dark sleep, spikes are driven by non-REM, without a doubt. This is not very unique because Rett syndrome kids will show the same thing. Most developmental disorders that have epilepsy this matches the profile that non-REM states make the brain more vulnerable for throwing spikes and seizures. And over here so this bar on the top is showing you the start of the EEG and end of the EEG but the light cycle on. So here the lights are off. Lights are on. Lights are off. So this the black portion adds up to 12 hours. The white portion adds up to 12 hours. And so here's the 0 to 24 hour indicating the duration of the EEG. This is the frequency plot for the number of spikes and you can see how they go up and down at P60.
So this is a younger mouse and then the same mice a little bit older. So in general you can see the frequency of spikes are going up but when the seizures happen, which are these red lines, when the seizures happened in the younger mouse there does not seem to be a direct correlation between when the spikes were happening and when the seizures because there seems to be a continuous occurrence of spikes. But then when you look at P120 you can clearly see what we have seen here with a non-REM and in the light cycle, there's a little bump during the light cycle which is this white bar here. So frequency of spikes is going up during their sleep during the light cycle and the seizures start occurring when this transition is happening. So as the spikes are going up the seizures are going up. So what all that could mean at this point is that susceptibility for spikes and seizures starts going up at that time point in the light sleep during non-REM. And this is just interesting to keep tabs on because as we start analyzing more of the EEGs coming from the kids with Syngap we will want to see whether this holds true for the kids too. And this has tremendous relevance for when an anti-seizure drug is given to the child. So let's say, just a very simple example, if this is a 24-hour cycle and obviously you're not going to wake up your child and give them medication. Well that's not usually the protocol. But if the seizures are happening at that time and you give your medication here versus here it's very easy to hypothesize that this dose given here maybe would be the early morning for the child will not be efficacious versus if you gave it here maybe it would stop all of these seizures. So I just want you to keep that in mind on if the seizures are occurring at a particular time of day but the dose and then the highest concentration of the dose in the body is at a different time of day that would for the same dose have completely different efficacies in the same child.
Okay so this is a hypnogram from a mouse. So this is what I mean by the... so here's, this is 24 hours, this is the light cycle. Here's dark and then the light. So daytime. Nighttime. Daytime. 12 hours each. And the same thing as we showed before. So the yellow is all the time that this mouse is awake. The blue is when the mouse is in non-REM and the red is when the mouse is in REM. So that's wake, non-REM, REM. So you can clearly see obviously there are differences between a mouse hypnogram and a human hypnogram but the stages are similar. The transitions are similar and when I said ultradian is that you see that because this is a light cycle that's when the mouse is sleeping and hiding away from predators there are many more sleep cycles. During the dark cycle when they are very active exploring, feeding, grooming, they're awake for most of the time but there are sleep cycles during the dark phase and then it moves again to the light phase. So basically you are now familiar with the three stages: wake, non-Rapid Eye Movement (non- REM) and then REM. I already said this, the mice are nocturnal and so they have the ultradian each sleep cycle. So hopefully now you understand what the difference is between a mouse hypnogram and a human hypnogram. So when we looked at these mice and their sleep cycles because sleep is a huge problem in many development disorders including SYNGAP1 here is a wild-type hypnogram. Here's the one of the younger the two-month-old Syngap mouse and then the same mice when they got older. And just eyeballing it if you see the same mouse at P60 versus P120 you can see the difference in how this mouse is now cycling. And when we quantified that, because mice are nocturnal and that is critical to their survival, if they start being active during the day and sleeping most of the time when they're supposed to be exploring and feeding you can see how that could be detrimental but when you quantify that you can see the wild-type there's a clear distinction of how much percent of the time they're in wake or sleep. At P60 this difference goes away a little bit but it's still significant.
At P120 this mouse has completely lost its nocturnal predominance of how it does its activities and that the same version, this is the same data quantified in a different way. So basically you can see for light and dark, red versus the blue, the wild-type, as you would expect a normal mouse, has a significant difference on how it cycles through in the light and dark cycle. And the P60, P120 this is lost to a greater extent. So this of course we have to see in the light of a mouse how this would be detrimental for the mouse's survival and well-being. So the take-home message from here is that the sleep cycles are completely disrupted in the Syngap mouse compared to their wild-type counterparts. And then here we have, this trace, you know, when the mouse is in the cage the video is on there's software where you can trace all the movements the mouse does during the 24-hour cycle. So this is qualification of once a wild-type and a Syngap mouse. Here's wild-type. Here's P120. And the software keeps tracing how the mouse moves. So you can see that during the day, during the 24-hour cycle the wild-type has this amount of movement. You can see a hotspot here which I would predict would either be where the waterspout is or where the food was. But look at the same so both this wild-type that is at P120 and look at the Syngap mouse. This tremendous hyperactivity. The hotspot is in the center here which would almost mean it's not associated with with where the food was or where the waterspout was. So it's just overall hyperactivity. And when you quantify that based on the light cycle where here's the the period when it's dark and this is normal so the if you follow the black line which is a control which the normal mouse does and so they are very little active during the day and when they are nocturnal their activity goes up and this is when they do all their movements, all their nesting, grooming stuff and then when the light comes on it goes down again. And then in the dotted line is the younger Syngap mouse and the solid red red line is the older Syngap mouse. And you can see there's some hyperactivity even during the cycle but during the night this activity is very significantly high and almost when the lights come on at the older ages they don't even stop. They keep going before it comes down.
So the take-home message here is that these mice replicate some of the phenotypes in the Syngap Syndrome where there's tremendous hyperactivity. Now I mean we will be doing additional tests to see whether this activity is directed or just not related to any task and here is the quantification for the things showing you the significant increase in hyperactivity in the Syngap mouse. Both at P60 there's a trend and then becomes very hyperactive at P120. So then we move. Now we transition. So now we know this mouse has seizures. We are excited that it has a phenotype of seizures that is actually seen in the patients. We are excited to know that they're happening at the transition points that our early human EEG studies are showing that they're occurring at transitions between wake and sleep and non-REM and REM. So like I said there's a huge area research of great researchers who are looking at gamma oscillations in the cortex because it underlies cognitive ability. If you are focused on a task your gamma oscillations in the cortex go up. Even if you're awake and you are not, you know, you're in a resting stage you're gamma oscillations will go down. So there's a very tight correlation of task engagement, cognition, learning and gamma oscillations in the cortex. Like I showed you before it's a 30 to 100 Hertz in mice because using protocols from our previous research we are looking at this 30 to 100 Hertz kind of gamma rhythm and then there's lots of literature to show that gamma oscillations are driven by interneurons. Now inter neurons are the neurons that are the "no-go" signal in the brain. So there's excitatory neurons which are the "go" signal and the interneurons that are the "no-go" signal meaning you stop the neurons from firing. And one class of these neurons is known as parvalbumin (PV). You will see this PV in all the next slides but it stands for parvalbumin interneurons and there's lots of literature to show that these interneurons they drive gamma oscillations in the cortex. So here's a simple schematic and I'll go through this again later on.
So what I just said that there are excitatory neurons meaning the ones that fire signals for us to do an activity if it's motor or to start a start a rhythm for certain functions in the brain and those are represented by these blue triangles. So the PYR is pyramidal neurons these are excitatory and they are in the cortex in huge numbers. Parvalbumin neurons it says GABA because that's the ligand that's released by these inter- neurons onto the excitatory neurons that gives them the "no-go" signal. So when this neuron is firing, if a parvalbumin positive neuron fires onto it (and that's why it's a "no-go") it will stop this excitatory neuron from firing. So that's very simple. Excitatory: go. Inhibitory: no-go. The red ones are the interneurons the blue ones are the excitatory neurons. But to make this slightly more complex, these interneurons are connected to each other with electrical synapses meaning they can then now fire together much faster instead of them communicating to one another but they also inhibit one another. So this is a very interesting circuit and this subgroup of interneurons is very critical in the cortex and we will go over this a little bit later. So for now right now what we're looking at is the "go" signal neurons, the "no-go" red neurons, they inhibit the excitatory neurons and they talk to each other. They inhibit each other and they also talk to one another. So that is the circuit in the cortex that many people have focused on because they underlie these gamma oscillations in the brain. So these PV interneurons are also called as fast-spiking interneurons and I pull this slide just so that people who have not seen firing patterns of these "no-go" signals can understand why this is a special class of interneuron. So here is patch clamp onto an interneuron and this B graph is showing you the single firing pattern of fast-spiking interneuron, which is our PV interneuron, a non-fast spiking interneuron, which are the other types of interneurons and a pyramidal neuron which was the blue neuron in a previous slide. So here's a fast-spiking. So this, if you take anything from this slide, is PV interneurons have a very high frequency firing way. What that means for electrical properties and how this neuron is built and is all interesting research that's ongoing and then you can see a non fast-spiking when it goes fast and then it peters out. And your neuron is a much slower firing pattern so very simplistically it would be if this blue neuron, which is an interneuron, has to stop this neuron from firing you would expect that it would have to fire it a much faster rate then the excitatory neuron. So this neuron would fire at this rate and stop this neuron from firing. And then the other (if I can go back a slide) the other characteristic of the specific parvalbumin interneurons is that they inhibit the neurons right on the cell body. It's like before the horse gets out of the stable it's a "no-go" signal. So it's a circuit which controls very tightly the firing pattern of the excitatory neurons. Other interneurons are known to form synapses on the branches of the parvalbumin neurons. So this is a very specific characteristic of a fast- spiking interneuron. And then we just talked about the firing pattern. So this is just to show you that these interneurons have the ability to spike at very fast frequencies compared to the excitatory neuron and other interneurons. So now this is the same data that we saw for the hypnogram before and we have color-coded it for the gamma power on the EEG for the mice. Okay. So the color code is still the same. We have taken out REM because in the mice they're very short duration. So basically this with the same color code this yellow color is the wake. Blue is the non-REM but instead of seeing the hypnogram now you're seeing every dot here is the 10-second EEG power for gamma frequency. Just for the gamma frequency.
Okay. So here's again the bar on top for the 24 hours. This is switched around for the way in which the data is shown. So it starts in the night. Here's the entire 12 hours of light and then here's the remaining 6 hours of dark. So what you can see in the wild-type, you can see this mouse is going through wake cycle, sleep cycles, wake cycles, sleep cycles but what is happening to the gamma power when the mouse is waking up and sleeping? As soon as the mouse wakes up the gamma power goes up. As soon as the mouse sleeps the gamma power starts falling. Okay. So this makes sense for the simplistic thing I said that when you are awake and active and doing something or reading something the gamma oscillations in your cortex will go up. When you sleep and as the delta slow waves start coming in, gamma power comes down. So this is a way how the brain adjusts and it's known that this is very critical for learning. So whenever the gamma power goes up and your're awake and you are learning a new thing, you're reading something new, it is very critical that your gamma power drops during sleep so that the brain can then put that what you've learnt or what you've read into your long-term memory. So that you remember it the next morning. Or with multiple readings in a few days. So that's how the brain learns or this is one of the components that's been proposed underlying learning and memory. So consistently, and this is just an expanded version of the data in this red box, here the mouse is awake. As soon as he falls asleep his gamma starts dropping. Even before the mouse is going to wake up, just as the child will wake up, the gamma starts going up. Okay. And you see that consistently. So you see the gamma oscillating up and down, up and down, during sleep. What happens in the Syngap mouse? So here is an example of the same analysis from the EEG of a Syngap mouse and you can see it's color-coded for sleep states but we don't- the oscillations become very muted and in fact if you watch closely here and here... here is the gamma during wake and it's actually jumping up during sleep. So it's the relationship of what gamma should be doing in the cortex is not only gone away but in certain cycles it's actually switching. So here you can see here's the gamma. It goes up during when this mouse falls asleep and actually goes down when it's awake and then in one cycle it actually goes up and so the relationship of the up and down that is so tightly controlled by the cortex is lost and that's what's shown here. So and in this cycle actually we're actually showing the gamma has gone out. So something has completely switched where the relationship of wake and sleep gamma oscillations, the high-frequency oscillations, is not only lost, it has reversed in certain cycles. And then we gave, like I said PMP stands for perampanel which is the AMPA receptor antagonist. So this was our first pilot study and and we gave a low dose which is 2 milligrams per kilogram and we just gave two doses. So this is acute dosing. Not long-term dosing to this mouse. And then the same mouse, this P120 mouse, when he got perampanel, it seemed like it started transitioning again normally. So here you can see this gamma and instead of going up in the same mouse it's actually going down. So now gamma oscillations have returned and that's why we call this as perampanel-mediated rescue of the sleep/wake gamma transition.
We were very excited to see this because now this is hypothesis driven right. We hypothesized that since Rick's lab has shown that AMPA receptor insertion is one of the critical functions of SYNGAP1 at the synapse that when it's not working and if there are excessive AMPA receptors what could happen to this transition and now if you give a drug at a low dose that blocks those AMPA receptors then the cortex now starts oscillating. Just like in control. So then we went in. We went and quantified this. So this is basically the wild-type like I said the dotted line is form wake to non-REM which would be that the gamma oscillations are dropping and the solid line is from non-REM to wake is when the gamma should be going up and so you can see here on an from the negative scale towards the positive scale and that's zero line as you would expect all the way to non-REM which is the dotted line or in the negative which means the gamma, you know, gamma power is falling these are slopes of the transition and then from non-REM to wake, when the cortex is getting engaged in gamma is going up, in the wild-type you can see all of those slopes are in the positive side and those two transitions non-REM to wake, are significantly as expected different from each other. What happens in Syngap this difference is completely lost and so that significance goes away. So whether it's one transition or the other, the brain cortex does not seem to be recognizing that it needs to switch to higher or lower gamma. When we gave perampanel, acutely, the same mouse we brought it back to quasi, very significantly back to control. So what we can take away from this data is the AMPA receptors seem to be playing a huge role in these transitions and that a drug that blocks those receptors can acutely switch it back to normal. Okay. And this is a very low dose, two milligrams is a low dose of perampanel.
Okay. What are we doing for time? Oh my god I almost took up the whole hour.
Mike: No, this is amazing please keep going.
Dr Kadam: Okay. So then of course I showed you the first Syngap child's EEG and we saw seizures even happening between non-REM and REM. So we wanted to look at those transitions. So we looked at from the yellow to the blue and the blue to the yellow. Now we will look at from the blue to the red and from the red back to the blue which is this and these are the heat spectrograms off the EEG and I just want you to just focus on this is non-REM to REM transition (this is in a wild-type) and if you just eyeball it you can see that even with a few second transition, the heat map off that EEG is changing. Indicating now the brain is transition from non-REM to REM. This is from a Syngap mouse and that transition now is not that self-evident. It's kind of blurred and when you quantitate that by power and I'm going to make this a little bit shorter by focusing on here for the gamma because we've been talking about gamma. The gray and the dark grey are the Syngap mice and and this transition is very weak compared to the wild type mouse and we give perampanel (the blue) it kind of brings it back and looks like the control. So these transition states by perampanel are being rescued both from sleep to wake and in during sleep from non-REM to REM. And that's what we've done. We've given a low dose perampanel. We've not done anything else and these are adult mice.
Okay, so that is what I wanted to focus on and we already talked about that. The other thing we did was we did because I just explained the importance of these PV interneurons and we know their role in gamma oscillations and when we had this finding but then beside we have the brains from these mice and that's why we you know we can take the sections, we can stain them and we should look at PV interneurons because there's lots of literature to show that PV interneuron or the dysfunction, drives gamma oscillation dysfunction. So we stained four PV interneurons which are in this fluorescent green that's PV. GluA2 which is a AMPA receptor subunit and then the blue which is just a nuclear stains with every cell will show up all the blue dots are all the neurons. The ones with a blue dot and the green are the interneurons and you can see they are far and few. So the very basic ratio of interneurons to neurons in the cortex is 1:10. So for every 10 excitatory neurons there's one interneuron and that's all the inter- neurons and then there's a subclass of those that are PV positive. So this is just a binary image of that on how we quantitate it and all these little puncta are the synapses that these PV interneurons are making on all these other blue cells. So if you just eyeball it you can see there's a significant difference between the wild-type and the Syngap mouse and so there seems to be a huge reduction in how these interneurons are branching, which we quantified here and you can see the PV counts which are the puncta, was significantly lower and the number of cells themselves was slightly lower. For the GluA2 we did not see any significant differences in the sense of how many GluA2 AMPA receptors were on these PV interneurons.
Okay. But when we looked in the barrel cortex, so the somatosensory cortex, we saw a completely different picture. Again the color coordination is exactly the same: the green on the PV interneurons, the GluA2 is the red, and because it's such a small signal here is a magnification and the arrows are showing you one specific interneuron (PV) and the expansion is showing you that same PV interneuron how much AMPA receptors it has on its soma which is the body of the neuron and this is a wild-type it has some, which we expect, but look at the SYNGAP1. It has significantly higher GluA2 AMPA receptors on the body of the interneuron, both in these are layers of the cortex two three and five six and this is iso cortical meaning similar to human cortex and so we saw this significant increase in GluA2. Now we gave pure perampanel which is a drug which would block these receptors and so the connection there is PV interneurons we know have a huge role to play in gamma oscillations and when we look at the somatosensory cortex of these mice they have this significant increase of AMPA receptor expression on their soma. So this is what I just said. GluA2 is generally expressed at very low levels and in GABAergic interneurons and there's previous work from other researchers and this this group is in Germany, where they artificially using genetic techniques did this right. It was not a Syngap mouse. They took a mouse and with genetic techniques did this to the mouse: increased AMPA receptors was on to GABAergic neurons. What happened when they did this the, GAD-GluA2 mouse, it significantly disrupted the gamma oscillation. So here is a mouse that they just increased the GluA2 on these GAD positive which is GABAergic neurons and it completely disrupted the gamma oscillation. So we are naturally finding this in our Syngap mouse but when somebody else did this using genetic techniques it resulted in the same disruption. So we were very excited when we came across this paper.
Ok so this is basically what I've been telling you guys about, what Rick's lab has contributed, which is a significant contribution and this is Yoichi's work showing very elegantly advanced and so this is a normal postsynaptic spine here are the AMPA receptors in red these little two things and there's a very very well control cycle of how many receptors are at the synapse, how they cycle, how they take it out of circulation and how they re inserted. Ok. So now SynGAP sits in this postsynaptic density and when it is not functioning which is this greyed out in this cycling gets disrupted and then there's no control and more and more AMPA receptors start getting inserted. Which is kind of- when I showed you the thing is this is what we predict is happening. So this mouse has no SynGAP in the postsynaptic and for some reason, specifically for these interneurons, more and more AMPA receptors get inserted onto the synapses in this cortex. So this had the data that Rick's lab has shown matches what we are seeing but specifically for interneurons. So coming back to the circuit that you have seen now if you put it into perspective that we know these interneurons inhibit this neuron right but we also know that these excitatory neurons synapse on to interneurons. That is how they get the signal. It's a feedback. When there's too much excitation the blue circuit goes and tells informs the interneurons okay you need to do something and then they come and stop. So there's input, the brain acts, the inter neurons stop the signal. So that's how the circuit is controlled very simplistically. Now what happens is as we have seen work with the immunohistochemistry and with Rick's data, what if now this excitation here becomes fourfold? Which means this interneuron will now keep getting signals to keep inhibiting the pyramid neuron. This you can see is simple in learning you would you would want some inhibition but not constant inhibition. This would almost be like a constant no-go signal and so that you can kind of try and understand how that would be for a cortex that's trying to learn something new.
But how is this relevant to seizures? People have done this experiment here. So what we just talked about is exciting GABAergic neurons, exciting these PV interneurons. So they use genetic techniques and, you know, the people used when you introduce a certain protein into specific cell types which is here, these green cells which are the PV interneurons and such that when you shine a blue light on them they all start firing. Okay. So this is a genetic technique and what they're showing here is here's the blue light here are there are green PV interneurons when you shine the blue light, all of them start firing. They start firing. What happens when you shut the blue light? So all of these interneurons are giving a no-go signal to all these interneurons 1, 2, 3, 4, 5. So that is here. So when the blue light is on they're firing altogether and we just saw how fast they fire. When they are firing there's not even a chance that any of these neurons can fire. So they're all silent these five neurons. What happened when they shut off the light, this group of these are also in the group. Shut off the blue light, this is called synchronous rebound spiking. Now all the neurons... they fire together! They're not supposed to! When they fire together what happened on the EEG? The mouse threw a seizure. So this is kind of reverse thinking where we think "oh, if the no-go signal is weak that's how seizures come on." What this group is showing if the no-go signal is too strong the brain can also throw a seizure. Now this concept has been around in the epilepsy field for many decades but now with newer techniques we are able to prove that this opposite way of throwing seizures is the possibility.
Now why is this important? I just showed you that there's so much excitation onto the PV interneuron and is that playing a role in these 3 Hertz discharges that the cortexes of the children and the mouse are throwing seizures for? So hopefully this is the summary so basically what I've shown you is we focused on cortical gamma oscillations. These are these low amplitude high frequency, and the wild-type transition high gamma, no gamma. The Syngap Mouse, no transition in fact sometimes it goes a little higher. You block AMPA receptors, it reverts back right away. Back to normal. So this is just the perampanel drawing. It's an AMPA receptor antagonist. So when you give the antagonist you can rescue that and these transitions are associated with these 3 Hertz discharges in the mouse. That's the same discharge in the human. So that's the human EEG and that's the graphic for that and then what I showed you was the immunohistochemistry arm showing as the way because it's the PV in wild-type and you can see the red around here which is here's the neck you know the black and white version of it of showing how many more GluA2 AMPA receptors there are on this interneuron from the Syngap brain versus the wild type. So what we this is denoting is that then this can become a vicious cycle. This hyper excitation can lead to this gamma oscillation disruption. This disruption in transition can lead to seizures. Now do seizure activity again lead to this further disruption in excitation? That would be an interesting question to parse out. So you've seen this before. Now you know you're familiar with what this means for the EEG of this child and so this is basically you summarize the 3 hertz short wave spike wave discharges. Seizures in transition states: wake to sleep, sleep to wake, non-REM to REM. Early morning seizures, which many parents report, the clinicians have reported, and then that would be a huge transition from gamma now suddenly has to go out which then it fails to most likely. Seizure clustering. Seizure clustering is important because if they're not just single events a bunch of seizures themselves can alter circuits and disrupt learning and memory in any child even in adult. So this basically summarizes everything I have said: the non-REM component, why we used perampanel. It was a low dose and how you saw just with a single low dose how it completely altered and that that is increased expression of GluA2 receptors in these PV interneurons.
So to summarize: SYNGAP1 patients I said 3 Hertz myoclonic seizures, the mouse has the same phenotype. Then children evolved with multiple seizure phenotypes we saw that too. Seizures in our transition states, we saw that. Low sleep efficiency we saw complete disruption of the sleep cycle in the mouse. ADHD is reported in the syndrome and then we saw the hyperactivity. What is novel, which usually people don't look at when they don't look at the background EEG in the clinical EEGs that are done in the children, is this abnormal gamma homeostasis meaning the transition between states for gamma oscillation. Progressive worsening of the epilepsy, which would be good to get from the Natural History/registry that you guys are working on and then this whole focus on GluA2. Now why is this important? Perampanel blocks all AMPA receptors. GluA2 is a specific subtype of AMPA receptors and currently there's no drug on the market that just blocks GluA2 specifically. So that would be interesting for us to figure out if, you know, to try and either use medicinal chemistry and screen for drugs that would be even more surgically like a scalpel rather than a hammer to block only certain types of AMPA receptors and then maybe we could get away with the side effects. Our future goals is where you know we are collecting EEGs, overnight EEGs with children who have been diagnosed with SYNGAP1 and then of course we're looking for different ages because we want to see how this is evolving in children as compared to what we've learned from the mouse. We are now also, we've amended our IRB study to include siblings. If there is a sibling who has or if you can get an overnight EEG for a sibling who does not have SYNGAP1 now that would be the best control for that background genetic background family for us to go to compare sleep cycles with the sibling versus the child with the mutation and of course we will keep testing novel interventions in the mouse that we have characterized but like I mentioned before we are now move to a much younger age and we're doing EEGs in pups. So here is the this is online and I will I will send these links this is the proof study at Hopkins and so basically it involves parents signing a consent form that I will send and if you have any questions like I'm happy to answer them and then what it requires for the parent to do is then sign a release form to whichever clinic or hospital the overnight EEG was done and then the hospital directly sends us the actual recording not the report we are happy to read the report but we want the actual EEG recording the 24 hours or however long it was if it's ambulatory EEG done at home we are happy to take a copy of that too. So that is the study we have been recruiting we have a few EEGs right now and now we're focusing on analyzing it now that we have this data from the mice and some understanding of what this EEG looks like and with that I'll close and acknowledge many Brennan who was is the first author on the paper and did the bulk of the work of the data I've shown you today and then all the people who have been in the lab who have contributed in multiple ways are funding and of course Rick's lab that we wouldn't be doing Syngap research if he had not invited us to join the team and with that I'll thank you for your attention.
Mike: Thank you Shilpa. If everyone wasn't muted I think you hear a lot of clapping and I apologize for being familiar right. I'm so humbled by what I just see I feel like I should call you Dr. Kadam more often but I've just been lucky to talk to you in the past so please forgive the familiarity. I know but you're awesome so I want to just make three points and then I want to point you to some of the questions in the chat. Okay. The questions have come from Hans and Marta who are both medical doctors and Syngap parents which is as well as two questions from Aaron who is the medical training as well and so they were able to keep up with you. I think the rest of us are still digesting but point one: you said sibling EEGs. That's actually not as hard as it sounds if you go to someone and say "hey, can I get a sleep study" right because a sleep study includes an overnight EEG so if my chubby little two-year-old is having bad sleep I can get someone to ask for a sleep study and there's a sibling for you.
Dr Kadam: But right but I mean it was are the parents of Syngap children who said that their neurologists for some reason and I'm really not clear on this, asked for or maybe because if they were twins they asked for the sibling the sibling without SYNGAP1 to have an overnight EEG and as a scientist that could be the because we would you know exactly what you said most of our control EEG is because we will have to age match because in children sleep cycles evolve. A three-year-old sleep cycle is not the same as the eight-year-old and so we are going to be getting our control EEGs from the sleep center at CHOP, Children's Hospital in Philadelphia and those kids go for this exact same reason you said that they have some of the child has some sleep problems no genetic disorders and then they get a diagnosis of no problems on the EEG. So we have a collaboration with the investigator there and once we have our cohort we will get age match ages from that Center to kind of compare blindly the Syngap EEG versus the control each year when I became aware that parents did have sibling EEG I actually took the trouble to amend IRB study so that we can also get a copy of the sibling EEG. Will it be very critical? I don't know. Once we have a cohort and we are able to look at it it'll be very interesting to look at the background EEG of the sibling because that would be the best control instead of a child unrelated child with maybe some other disorders that might affect that child EEG but that's the best control we can get so whether it's easy or not is you guys will give me feedback on because I think it happen organically and I found out it's happening I don't know how easy or difficult it'll be for you to go and actively seek sibling EEGs for your children.
Mike: I'll let you know but the second point and I think the takeaway that anyone who enjoys this presentation will walk away from this is they're gonna do what I did after I read your paper and walk into my neurologists office and say "hey, could we try perampanel?" and my neurologist says well, there's a lot of side effects and blah blah blah and then I say yes, but what this paper suggests (and please tell me if my interpretation here is wrong) what this paper suggests is that actually perampanel at the dose that it's currently recommended for is an AED yes, we know has side effects, but this paper suggested at very low levels where maybe we could avoid the side effects perampanel could have an impact that's my first question to you my second one is I just want to draw a thick line under something you said earlier where you pointed out that the same drug at the same quantity at a different time of day yes to the seasonality or whatever the right term is of our kids yours can have radically different impacts and I've seen that just in one of the sleep through that we give our kid guanfacine for sleep and moving that from but we gave it to him for behavior but it turns out to help sleep we move that from morning to evening everything changed. So I just want to emphasize that point you made because I've lived it but the question really is about the perampanel. Is it true when we're talking to our neurologists about this but we are talking about radically lower dosing that is currently indicated at for perampanel as an AED is that right?
Dr Kadam: A hundred percent. So the current protocol for introducing perampanel to any child (or any adult) is to start with the low doses and then they'll ramp them up over a few weeks and then it goes to they may not even start at 2. If they start at 4 the goal is to reach up to 12 milligrams per kilogram. So that is a substantially higher grade of perampanel dosing. But the major point is: if you talk to any parents I've talked to and very few Syngap children have actually got perampanel (that I'm aware of) but other syndromes where perampanel has been tried this is the common complaint that there are multiple side effects. But there's a distinction: there are no side effects when the low dose is on. The side effects come on when they ramp up the dose and then usually people will just stop it. Right. And that's understandable if you do it in an outpatient and you tell your clinician down and now this there's a child sleeping a lot or whatever the problem is with the perampanel dosing. So that's a consistent story. What our data is saying because ours it is so specific let's say, you know, these interneurons are showing this high AMPA receptor, right. So what is the logic behind that? Why is the low dose working or why we think it's working? That only with the low dose. The side effects come when the high dose block most activity right? That's where the sleepiness comes in and all the side effects come in.
Why is the low dose working here? There are few cells as I just showed you that have this very high level of AMPA receptors. So if you gave a low dose that same brain what would that the maximum effect of the low dose would be on the cells that have the highest AMPA receptors. Not on all the other cells and so the understanding would be that you could get away with the low dose which has no side effects but have the most highest therapeutic effect on the cells that have abnormally high level of AMPA receptors and remember Syngap is the only one that I'm aware of as a development disorder where the specific pathology of high AMPA receptor insertion is understood. Thanks to Rick's lab. Most of the other epilepsy there's in general high excitability which they given perampanel to block the entire brain from seizing so here perampanel would almost act like a scalpel instead of a hammer it could be for us it would be even better if we found a drug that was only GluA2 specific which does not exist (but might in the future) and the idea is absolutely right then that this paradigm does not exist for neurologists right now they only understand the start to the low dose and ramp up.
For a me as a scientist it's very clear that the side effects are coming when the ramping up happens not when the drug is just given in the low dose. So that's one thing to consider. And two: if the seizures are coming because of the disruption of gamma oscillation then maybe (and we will test this out) maybe just a low dose perampanel is enough for the Syngap child. It may not be true for any other syndrome it's very specific to the SYNGAP1 mutation. The second important point you said was when is this drug being given? So right now Syngap is given you know usually twice a day so you usually with an 8 hour difference, sometime in the morning, sometime before bed. What if we as we look at more EEGs and we figure out that these transition points are when the child is most vulnerable and... now if you look at all the transition points I talked about is wake to sleep, sleep to wake, REM to non REM. All these are now tied to the sleep cycle. So if the only dose if it's given only once a day is given right before sleep and then your biggest side effect is sleepiness then you counter both. It's a win-win. And then you don't give it in the morning... and this is all hypothetical right now okay, so I'm just talking about the fact that what we are seeing and how the drug would be best given. This has to be parsed out in the clinic. So those are the two times yes the time point when you're giving the drug. What are is known side-effects? When are you expecting the most seizures to happen in the child? And then why would you give it in the time when it's not needed? So those are the points of things that I'm hoping as we start talking about this data with neurologists is changing paradigms and protocols in clinic is very difficult. People do not... and I'm not even aware how this ramping up dosing protocols came into place like who tested it out how was it tested out and you may know that Takeda now is actually testing out the use of perampanel now in children less than two years of age for safety. As a safety trial. So it's moving in the right direction. These two points are very critical. Why would you ramp up if, based on the pathology, a low dose would be actually the best way to go? And then secondly, why would you give it multiple times a day if you want to just focus on the most vulnerable time when the brain is seizing? So those are the two things.
Mike: Thank you. I know we're over time but people aren't going anywhere because I think they're also enthralled. Have I forced muted everybody? Should I unmute you Martha and Hans and Aaron so you guys can ask your questions? Here Hans I'll unmute you. I'll try to - it's not working. Martha are you muted?
Marta: I'm here, yeah. I have to use a different one because the connection is bad. My question is: did you notice any difference on the behavior of the mouse after with the Fycompa? Did you notice that they have the activity improve or something?
Dr Kadam: So that's what we're doing now. We're actually... we are going to start looking at not only how their behavior changes but how their behavior is behavior testing for cognition sure what we're going to see but now we know what we expect to see now questions to be asked.
Mike: Thank You Hans, Aaron do you want to chime in with your questions like one to two seconds with like a 3.5 to 4 Hertz frequency oftentimes the colleges are you observing those same kinds of patterns in the mice and then also would you classify those as seizures as well?
Dr Kadam: that's a very good question and and that brings us to the point of just animal modeling so that's why I mentioned what the similarities were but obviously a mouse is not not a child and we do when I said short duration myoclonic seizures they are sharp but they are not like 1 or 2 seconds long. We do see one or two second long seizures that are not the typical spike. I showed you the one where the EMG was completely silent. So in that sense it's not the exact phenotype which by the way there is no such mouse model of epilepsy where you have the exact human phenotype especially not for genetic epilepsy. The closest we get is with temporal lobe epilepsy where there's very high seizure frequency and there's only one type of generalized tonic-clonic seizure but coming back to your more important question is when neurologists, when there's a short duration discharge and that's not classified as a seizure that's just the nomenclature and it's important for classification in the clinic and the reporting but as a neuroscientist, any epileptiform discharge we know, even a single spike, people have shown disrupts the learning memory circuit in the hippocampus. So whether it's a one second or two second discharge versus a four second discharge for me as a scientist that's still important to identify and quantify and human EEGs really do not go into that level of granular... it's almost ironic that we use much higher resolution and granularity when we quantify the mouse EEG but the similar thing does not happen even with these overnight EEGs when there are multiple seizure events. Like, for example, the work, the quantification and the trace I showed you for the one child. The report did not say how many events happened or where they happened. We had to go and identify them and clock the time and superimpose it on to the underlying power and the sleep EEG. So basically to shorten that answer: yes in the clinic a short duration epileptiform discharge is not classified as a seizure band but for a neuroscientist if a normal brain is not having that kind of episode we know for a fact even short duration discharges can disrupt circuits and that's why it's important to us.
Hans: Can you hear me okay? Yeah. Great, thanks so much for the great talk. I just had a question because I believe Gavin and maybe Jimmy had a paper showing a diminishment in interictal spiking when SynGAP expression was sort of turned on I believe with a floxed Mouse. Is interictal spiking something seen universally in our in our children or is it something hit or miss?
Dr Kadam: That's a very good question. So we have not started to quantify just spikes in the EEGs. We are still collecting. Once we have a good cohort and that's why we are urging parents to send us, or participate in our IRB study. For sure the report does not tell you how many interictal spikes there were. Even in the one that- so far the ones we've seen I have not noted a lot of interictal spikes. Yes, we've seen the seizure events but although they are the 3 Hertz seizures that I was trying to correlate between the mouse, they're not exactly the same and the short duration high frequency 3 Hertz spike wave discharges that are commonly reported even in the reports are very unique but they're not unique to send get and I don't know even from the literature how many of when when the epilepsy phenotype is predominantly 3 Hertz spike wave discharges how many how what what role do in trickle spikes play in that epilepsy and that that literature is not that strong I would assume it and it's never come my way and even for the few EEGs we have looked at from the clinic intraictal spikes of course they occur but almost not at the frequency at which we see it in the mice and this is I mean this is pre and this is premature at this point because we have not yet looked at multiple EEGs and so there it'll be interesting to see whether certain children who have the same phenotype but have a lot of integral spikes that'll be very interesting so both yes that is important point to look at the ones we've looked at we don't have a very high predominance of intrical spikes but that does not mean that there aren't send your children but there's lots of literature to show because they are people are using Medtronic companies that are implanting units onto the scalp of patients is you are using a Gotham's to quantitate interictal spikes to be able to predict when that adult is most likely going to have a seizure. So the idea being that then the patient will be injected with the drug or the or the anti drug into the focus of wherever the seizure is starting and that's that's a new way of therapy for in temporal lobe epilepsy or cortical lesions so that's where interictal spike research is very very strong. It's it's strong because it's being used as a predictive marker for oncoming epilepsy but in developmental disorders spikes do happen. In Rett syndrome in their EEGs and they always come in non-REM. What they mean for seizure prediction is an interesting question that I don't think people have yet started looking in very young children. Does that answer your question?
Mike: He's muted. Yeah. Hans?
Hans: It sounds like it remains to be seen based on a survey of child, Syngap child EEGs, whether or not they have comparable interictal spiking.
Dr Kadam: Correct. Correct. Okay great. And in Rett syndrome we know there's massive interictal spiking. I'm not seeing that- yes once it's characterized in the clinic that'll be very important. It will give us a lot of important insights.
Hans: Great, thank you.
Mike: And so just conscious of time, next steps we're gonna share out the recording of this pretty quickly and then Dr. Kadam, if you can share the link to that study you're doing we will make sure to push that out as well.
Dr Kadam: Yeah I'll send you an email right there this is done both I'll attach both the forms and you can review them if you have any questions please feel free I'll send my email address too and and we are, we are trying to collect these overnight EEGs.If you have multiple, in the sense that your child when they were three they had one and then they had one again when they were six that would be priceless for us because then we would have a temporal progression EEG for the same child and then if you guys have a you know one if you guys talked you know and figure out whether sibling EEG is something global please let me know and also let me know how I can help.
Peter: Dr. Kadam, just to follow up on that question, if we have an EEG from after the child is two years old and one from just before they're two years old overnight EEG would that still beneficial? Yes it's only unfortunately because of how the IRBs were initially written and special forms need to be filled when the child is less than two years old I will have to write the amendment to get an EEG that's from a child is less than two years old. So right now we are approved from two years old to 16 years old but I'm working on the amendment so at this point if you have one that's over two years old you can send that to us and then once the amendment is done the consent form will already be with us I'll just send you the updated amendment and then you can send me the earlier EEGs.Sounds good. Which is, I mean I don't know but it's very interesting for us as we're finding that there is one subgroup of children that is having seizures and severe serious very early on and the registry and the clinicians who are working on this better they characterize this the better it will be for the field moving forward because definitely something that's happening in the brain that's less than two years old it's a whole different ballgame then something is happening at five years old and of course that the class of drugs changes because most drugs are not approved to be given for that younger child. So then that's why management becomes different and then so then the science needs to match what's happening at the bedside and so then we have to start thinking that way. The more we know the better we will do.
Mike: Marta any other questions before I wrap it up?
Marta: My question is about the age. With my daughter she's 17 she's one of the oldest that then connected then you said it's 16 years old...?
Dr Kadam: So if extending it beyond 16 is not a problem because out there there are no extra rules and regulations with the two-year-old child we less than two years old we have to fill these extra forms so now if I'm going to do the amendment is that the only EEG she has is after 16?
Marta: No no no, she has, I actually had several of them to send you. The other question is: she's probably one of the few that doesn't have any seizures during EEG. Or at least reported.
Dr Kadam: And you see...?
Marta: She doesn't have any activity reported and then but it still theta. EEG is abnormal they're not...
Dr Kadam: 100% so I mean those are some of the most valuable EEGs because we know that seizure events also have effect on these background oscillations. So if there is a recording of EEG where it is not a single event that will also provide additional insights and also like I said, these seizure events most likely also are dependent on the age. So there may be ages where the child is more vulnerable to higher seizure events and there may be "honeymoon periods" as have been shown in other development disorders and we don't know what that is for SYNGAP1 and what is the range or whether there are specific stages and whether all children fit in that stage or there are mostly outliers. All of this information hopefully now will start trickling in as more and more people start getting involved.
Mike: Can I...? So I'm gonna wrap it up unless anyone's got a burning question. I want to thank everyone especially the people from the Kadam lab for their time. Do you mind going back quickly to slide 8? I just think this is... I thought that was a super cool slide but I didn't want to interrupt you. That diagram is amazing because I think that's what we live through right? We have young kids the P60 who were only having myoclonic seizures and the parents aren't sure and they go to doctors and they go to neurologists and they take videos and people say they're not sure and it takes time and then a few years later, proxied by the P120's, we have kids who are sure having seizures and we start seeing different phenotypes. So point 1: I just think this slide really as a Syngap parent and someone who talks to Syngap parents every day really speaks to me and I think it's awesome. That's point one. Point two is I just want to highlight for the parents generally, this is what we- this is why SRF exists right? To connect parents and scientists. To emphasize the great work that scientists are doing. There's a lot of names on that last slide but Dr. Kadam has been someone who's taken our calls and talk to us many times. Brennan who's the first author on that paper is someone who was at our round table last year and as a young researcher and someone we're excited to see stay in Syngap for a long time. So this has been a great webinar and I'm so grateful and the third point is just to build on that and say thank you. This was great. This paper's amazing. Hopefully we'll have more kids on perampanel. I think offline will ask you about why we wouldn't just do a study. But thank you so much for the work you do, for your lab, for this paper and for doing this webinar. Thank you.
Dr Kadam: Thank you guys and thank you for the opportunity. I mean the feedback from parents has been one of the biggest motivation for my group for Brennan for sure and and yeah and for us as as researchers getting inputs of what's happening in the life of the patient and even in the family because quality of life is an important component of us understanding. You know "cure" is one thing but there are many ways in which you can improve categories of the disease and so although we can keep cure on the horizon as the goal where we are headed to, we cannot miss all these side routes where we can make substantial improvements in the management and understanding of how the disease is progressing and that only comes from communication so thank you guys for this. Thank you so much that was very good. Thank you I appreciate it. Thank you. Bye bye guys.