Clinical and Research Advances in Non-Epileptic Seizures Seminar

Because the signs and symptoms of non-epileptic seizure (NES) can resemble epileptic seizures, patients are often empirically treated for presumed epilepsy and receive anti-seizure medications and invasive treatments, which do not treat NES. Up to 20% of patients diagnosed with epilepsy have NES, making this a common condition, frequently seen in neurology clinics and units.


You will hear

This seminar described research advances in the diagnosis and treatment of patients with NES, including biomarker studies examining data from bioengineering and from structural and functional neuroimaging.


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About the Speaker

W. Curt LaFrance Jr., MD, MPH, is Director of Neuropsychiatry and Behavioral Neurology at Rhode Island Hospital (RIH) and Professor of Psychiatry and Neurology at Alpert Medical School, Brown University. He is Staff Physician at the Providence VAMC, and Clinical Lead for the VA National Telemental Health Center Tele-Seizures clinic. His research focuses on developing new biomarkers and treatments for neuropsychiatric aspects of epilepsy, conversion disorders and TBI.

This seminar is part of CURE Epilepsy’s Frontiers in Research Seminar Series. This program is generously supported by the Nussenbaum-Vogelstein Family and aims to help educate and expose researchers, clinicians, and students to exciting epilepsy research and also provide opportunities for young investigators to interact with leaders in the field.

Q&A with W. Curt LaFrance Jr., MD, MPH

If non-epileptic seizures reoccur, are epileptic seizures are also likely to reoccur?

We follow people over the course of time, and what we found, interestingly, is there can be periods… what we call periods of quiescence, or quiet periods, where there’s no seizures. This can be for epilepsy or for non-epileptic seizures. In taking the history, in talking with people, we’ll say… They’ll come back, and they’ll say, oh, I just had a flurry of seizures again. This could be epileptic or nonepileptic seizures. The question is, anything different recently?

More times than not, what we’ll hear is actually, I was getting ready for a final, and I pulled an allnighter. And then after the final, I had the seizures again. Non epileptic Seizures Diagnosis, Treatment, and Strategies Page 13 of 22 Again, this could be for epileptic or for non-epileptic seizures. As you’re aware, sleep deprivation is one of the ways that we actually induce seizures, whether epileptic or non-epileptic seizures. So, I’ll see what we’ll call environmental changes. That can be after a period of a quiet period, and then a flurry again, or recurrence, sometimes environmental. Sometimes it can be physiologic changes in the person. I see this from kids, to adolescents, to adults, I see this with other medications being added, bodily changes happening, all kinds of things.

There were some questions that came in, asking about whether there could be age-related, from childhood to adulthood, can that induce more nonepileptic seizures? There was also a question about whether trauma, for example, having wisdom teeth taken out, can that induce? That’s a pretty big stressor in a young person’s life.

Yes. You said a very important word there, you said stressors in their life. So, I’ll hear this from some people, they’ll say, but doc, I wasn’t even stressed, and I had the seizure. So, I’m not talking about stress-induced seizures, I’m talking about life stressors contributing to the formation. What I mean by that is, sometimes it’s not in the ramped-up period, where a lot of things are happening in life, or somebody just had a procedure, or a surgery, or something like that. They do happen postoperatively. I’ve seen them coming out of anesthesia, nonepileptic seizures.

But also, after procedure, like wisdom teeth extraction. But it also happens in what I call the letdown period. What I mean by that is, I’m on the beach, and I’m with my family, and it’s wonderful, and I had a seizure there. What’s that all about doctor? So sometimes people, when they’re in the ramped-up stage, they’ve got their defenses up, and then they’re when they’re in the letdown stage, then their defenses are down, and that’s where the seizure may occur. So, there’s not a one-to-one relationship of, I was stressed out, I had a seizure. It could be in different types of environments. So, I refer to life stressors, life events… we’ve all got life events.

Another thing that people hear, is that well, we’re not seeing an EEG signature, but it’s because the seizure is occurring deep in the brain, and we just can’t pick it up. How would you respond to that??

There are some locations, or foci, in the brain that elude scalp EEG signal. So, what I mean by that is if you’ve got a what we call mesial temporal, or some frontal lobe seizures, epileptic seizures, they actually… you can have the epileptic seizure, and the scalp EEG is not going to pick up that abnormal brain cell firing, epileptiform activity. So, in that case, it’s not that it’s not epilepsy, it’s that the focus eluded the scalp electrode. So we’ve got a clue there, though.

So just because you haven’t a normal EEG doesn’t mean it’s not epileptic seizures. We’ve got a clue, though, and we use the term semiology, ictal semiology, and all that means is the physical characteristics of the seizure. There are certain ways that frontal lobe epileptic seizures present, characteristically, that differ from psychogenic non-epileptic seizures. Even though both of those might have scalp negative EEG, we can look at the ictal semiology, the physical characteristics of the seizure, and we can make a comparison.

So that’s why you heard me say earlier, the right history, with the right witnessed seizure, with the right EEG, those are the ways that we get the documented… That’s how we get documented non-epileptic seizures. If we have the seizure characteristics, even though it’s a scalp negative EEG we may say, hmm, this looks more like frontal lobe epilepsy than it does psychogenic non-epileptic seizure, just because I’m watching the seizure myself. That’s the importance of the video EEG.

How do we find providers were trained to provide these therapies in our states or regions?

I would say start with your local epilepsy center, and say, do you have people who are trained in treatment for non-epileptic seizures? Now, that doesn’t mean that they were trained with taking control of your seizures workbook, they may have their own approach. What I showed you was just one of a number of approaches. So, this is not the be all and end all for everybody. There may be places around Non epileptic Seizures Diagnosis, Treatment, and Strategies Page 15 of 22 the country, who say yeah, we’ve got somebody who’s been treating people for 20 years, and they use this approach.

So I would say start with your local seizure, with your local epilepsy center, your local epileptologist. If there’s a neuropsychiatry department, sometimes they’ll do overlap brain and behavior, and you can contact them. I would say start locally.

Are non-epileptic seizures ever a diagnosis that can be removed from a patient’s problem list? This is something that needs to be on the radar now and indefinitely.

I view seizures, whether epileptic or non-epileptic seizures, as a chronic medical illness.

I’m thinking now of the International League Against Epilepsy’s more recent definitions for epilepsy. Before, there wasn’t a great definition for resolved, but now there’s a category for resolved epilepsy. So just as there’s a category for resolved epilepsy, you can have a category for non-epileptic seizures. That’s not official from the ILA per se, but I’m thinking, in parallel, what’s been done for the new diagnostic criteria for epilepsy and terminology, that could also be done for nonepileptic seizures.

Here’s what I will say. Life events are still going to keep happening, life is going to keep happening. Somebody is going to get sick in the family, a bill is going to come due that you didn’t expect, the car is going to break on the day that you don’t want it. That’s always going to keep happening. The tools that patients get with the workbook; they have to keep applying those tools.

The way that I demonstrate that to patients is, if they have readers, then at the end of the treatment, I’ll say, okay, read this sentence, and they’ll read it. And I’ll say, now take off your glasses and read the next sentence, and they can’t read the sentence. And I’ll say, the glasses didn’t cure you. You have to have the glasses on for you to be able to read, you have to keep using the tools for you to be able to address the stressors, the ongoing life stressors.

So people can go for extended periods… They’ve come back two or three years later, and they’ve said, the stuff came back. And I said, how are you doing… what’s going on in life, and how are you using the tools? And they said, life has gotten a lot harder, I just had two kids, and I’m not using the tools. I forgot about. So, there’s a little booster, and that booster is the thing that helps them to get back on track to use the tools again to address life events that are going to keep happening.

A couple of people have asked about the role of psychogenic seizures, non-epileptic seizures, and posttraumatic stress. I think you’ve already touched on this a bit. It’s not just the current life events that are happening, but the sequelae, as well, and using these tools to address that. Correct?

Yeah. So, people will refer to the various comorbidities or cooccurring illnesses. I didn’t put the slide in, but you’re probably already familiar. We’re talking about epilepsy now. Anywhere from a third to a half of patients with epilepsy also have depression. Anywhere from 20 to 40% of individuals with epilepsy also have anxiety. Anywhere from a third to a half have cognitive issues with epilepsy. So, these are comorbidities, neuropsychiatric comorbidities that occur with epilepsy, very similar in non-epileptic seizures.

So you’ve got about half of the people have comorbid depression with non-epileptic seizures, about half have anxiety, about half of… 40% of civilians, and up to 70% of veterans have PTSD, as you would expect, with non-epileptic seizures. So, a lot of comorbidities. So, it’s not just about treating the seizure. That’s why I was saying earlier, you’ve really got to treat the whole patient.

Can there be a false diagnosis of non-epileptic seizures? Why and how?

The answer is yes, and it can go either way. You can be diagnosed with non-epileptic seizures, and it can be epilepsy, or you can be diagnosed with epilepsy, and it can be non-epileptic seizures. I’ve seen both of those. I’ve mentioned with one of my early distance mentors was Orrin Devinsky, and he said early on, Curt, you have to approach a patient with seizure with humility, because you can be fooled either way. So, people can say, oh, well, I’ve seen that ictal semiology, I’ve seen the physical characteristics of that seizure, and that… That’s got to be a pseudo seizure, they said in a dismissive manner.

You know what? Number one, it’s not a pseudo procedure, and number two, it was actually a very odd manifestation of a frontal lobe epileptic seizure. Conversely, you can have somebody it’s like, wow, that’s a story for epilepsy, I’m going to treat them for two or three years with anti-seizure medications for presumed epilepsy. Nope. This was not epileptic seizures. The AED’s are not going to help the individual.

Do none-epileptic seizures present during sleep?

The answer is yes. The devils in the details here. So, epileptic seizures can arise out of physiologic sleep. Non-epileptic seizures can occur during the nighttime when a person is sleeping. Those are two different statements… So, both of them can occur at night, both of them can occur while people are sleeping. But what I mean by that is, we look at the tracing on the EEG. As many of the individuals know, the EEG changes when we’re in sleep and out of sleep stages.

So what happens is, we’re watching somebody, and then there’s an arousal, so they become awake, out of sleep, and then they have their non-epileptic seizure. That’s how we see that. But we really need the EEG that corresponds to the video to be able to say, oh, you know what, there was an arousal, so they were awake, even though it was at nighttime, when they were sleeping, and it was a non-epileptic seizure, as opposed to literally coming out of physiologic sleep and treated to a seizure. More times than not, it’s going to be epilepsy.

Do you have to have regular events in order for them to be classified as non-epileptic seizures, or can very infrequent events still be characterized as such?

I’ve seen people who have them once a year, and I’ve seen people who have 30 in a day. So, I wish it was that simple, it would make my job a lot easier. But no, it’s never that simple. So, we really have to pay attention to how often are these occurring? When are they occurring? In the workbook, we have a thing called a seizure log, and that’s where the individual really pays attention to their symptoms. So, every day, they’re documenting, I had one seizure at 12:00 PM, 12 noon, in the kitchen, after I was preparing breakfast, and it had this effect on me. So, we get them to start paying attention, whether it’s an epileptic or nonepileptic seizure.

And then when they start to pay attention to what’s happening, and what might be a precipitant to the seizures, then they can use some of the tools to go back and say, when I have my aura, number one, I want to get to a safe place, first thing, I want to let somebody know, if I can, if I have the ability to do that, and then I want to use some of the tools that I’ve been learning to
apply, to, hopefully, prevent the progression of the seizure into the full blown seizure. We’ve seen people with epilepsy and with non-epileptic seizures be able to take that approach.

If there’s nobody in the area in somebody’s region that’s an epilepsy specialist, what can they do? What can this person do? How can they find either remote resources, or is that possible? There are there lots of people in rural areas, or unable to get to an epilepsy center and see a specialist. What do they do?

They keep being an advocate for themselves. Here’s what I mean by that. I tell people, my patients and family members, you are your best advocate. I’ll hear statements like… sometimes they’ll say, doctor, can you write me a letter, I’ll say I Non epileptic Seizures Diagnosis, Treatment, and Strategies Page 21 of 22 can, but I want you to write the letter, and I want you to write the letter to your doctor, your hospital, your congressman. I want you to write letters to the licensing boards. The reason why I say this is because I live in two different worlds on a number of fronts, so in neurology and psychiatry. I live in two different worlds at the same time. In the VA and in the civilian world, I live in two different worlds at the same time.

So interestingly, with the VA system, being trained in telemedicine, I can treat veterans around the country. I go to my office in Providence, Rhode Island, and I see veterans all around the country, because the VA is a national system. So, one of the reasons that people can’t treat across state lines is because for me to treat somebody in Georgia or Arizona, I have to have a license in the state of Arizona, and I’m not going to get 50 licenses to be able to do that. So that’s why I’m saying, lobby. Go to the boards and say, you know what, telehealth has been helping people around the country and around the world, let’s make sure that people who have specialties can treat people, or people who are primary care can treat across state lines, and not have that burden of the system that exists in the civilian world. That would be a way to really push the envelope, which I’m a big fan of.

So that’s what I would say, keep being an advocate for yourself, but also keep asking around. A lot of times people will say, well, I’ve got a local clinician, they’re familiar. I view us in medicine as eternal students. So, some people will say, I’ll see you, and I’ll read the workbook, and we’ll work through this together. That’s another option, is have the local people, whoever it is, to get equipped using the resources that are available. Keep advocating for yourself, is what I would say.

Are non-epileptic seizures less dangerous to the brain than epileptic seizures?

People will ask, am I going to get brain damage from these? What I will say is that… sometimes people who have certain types of epileptic seizures, they can drop their oxygen level, and they can become hypoxic, and that can actually affect the brain, as you’re aware, we don’t see those same oxygen level drops in people who have generalized tonic-clonic non-epileptic seizures. So over time, we don’t see the same brain energy risks that may be associated with some types of epileptic-seizures that we do with non-epileptic seizures.

Serotonergic Mechanisms of Seizure-Induced Central Apnea Seminar

Respiratory dysfunction following generalized convulsive seizures is proposed as a causal mechanism in SUDEP. Central chemosensitivity to carbon dioxide, or CO2, drives ventilation in response to hypercapnia, especially during sleep. Dr. Richerson and colleagues have found that this response may be impaired in people with epilepsy, and that convulsive seizures lead to prolonged inhibition of the ventilatory response to hypercapnia.


You will hear

In a mouse model of Dravet syndrome, Dr. Richerson’s team has obtained evidence that seizures inhibit serotonin neurons that detect changes in CO2 levels. Defining the mechanisms of seizure-induced central apnea may lead to novel preventative treatments for SUDEP.



About the Speaker

The seminar was presented by George Richerson, MD, PhD, Professor and DEO/Roy J Carver Chair in Neuroscience at the University of Iowa, Carver College of Medicine.

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This seminar is part of CURE Epilepsy’s Frontiers in Research Seminar Series. This program is generously supported by the Nussenbaum-Vogelstein Family and aims to help educate and expose researchers, clinicians, and students to exciting epilepsy research and also provide opportunities for young investigators to interact with leaders in the field.


Understanding Post-traumatic Epilepsy and Impacts on Quality of Life

Dr. Pugh described the association of lifetime mild traumatic brain injury (mTBI) history, epilepsy, and post-traumatic epilepsy (PTE) with quality of life outcomes in post-9/11 veterans. Dr. Pugh’s work uses survey data as well as data from the Veterans Health Administration. Worse quality of life was found for persons with PTE vs either epilepsy or mTBI alone. This research demonstrates the impact of lifetime TBI and the importance of measurement of health outcomes in epilepsy. 

MARY JO PUGHAbout the Speaker
The seminar was presented by Mary Jo Pugh, PhD, RN, a retired Air Force nurse, and Professor, Department of Medicine, Division of Epidemiology at the University of Utah School of Medicine. She is an investigator at the Informatics, Decision-Enhancement, and Analytic Sciences Center of Innovation (IDEAS COIN) and the VA Salt Lake City where she leads the COIN focus area that aims to identify and mitigate health risks in Veterans across periods of vulnerability. Over the past decade, she has targeted her work using VA data to identify phenotypes in populations with complex comorbidities such as those with traumatic brain injury (TBI) and complex multi-symptom illness in Gulf War-era Veterans. Dr. Pugh’s current work related to TBI includes longitudinal observational and prospective studies to identify the emergence of distinct neurodegenerative conditions such as cognitive impairment and epilepsy and clusters of multimorbidity that may have similar or networked biological underpinnings.

This seminar is part of CURE Epilepsy’s Frontiers in Research Seminar Series. This program is generously supported by the Nussenbaum-Vogelstein Family and aims to help educate and expose researchers, clinicians, and students to exciting epilepsy research and also provide opportunities for young investigators to interact with leaders in the field.


Understanding the Mechanisms of Epilepsy in mTORopathies (FCDII and TSC)

Hyperactivation of mechanistic target of rapamycin (mTOR) signaling is implicated in a number of focal cortical malformations associated with intractable epilepsy. While the link between focal cortical malformations and epilepsy is well known, the underlying mechanisms remain unclear.

Dr. Bordey focuses on the role of hyperpolarization-activated cyclic nucleotide-gated potassium channel isoform 4 (HCN4) as an mTOR-dependent driver of epilepsy in tuberous sclerosis complex (TSC) and focal cortical dysplasia II (FCDII).

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About the Speaker
The seminar will be presented by Angelique Bordey, PhD, Professor of Neurosurgery and Cellular and Molecular Physiology at Yale School of Medicine. She is member of the CURE Epilepsy Scientific Advisory Council and a former grantee.

This seminar is part of CURE Epilepsy’s Frontiers in Research Seminar Series. This program is generously supported by the Nussenbaum-Vogelstein Family and aims to help educate and expose researchers, clinicians, and students to exciting epilepsy research and also provide opportunities for young investigators to interact with leaders in the field.


Q&A with Dr. Angelique Bordey

The resting membrane potential in the Rheb neurons, was it normalized during your current-injection test?

Dr. Bordey: Whether it was holding the cells at the resting membrane potential, which we did to inject. We held them at the resting potential and then injected current-injection. Like it would be more physiological condition. I’m not too sure it was a proper answer to the question, but I see it. They are depolarized compared to controls.

Do you see HCN4 staining in all FCD-two patient samples or only in a subset of samples?

Dr. Bordey: So far we saw in all of them. Yes, in all of them. We do not know the gene variance for these FCDs. As I know there are 30% or more FCDs that are due to mTOR gene variants, I don’t know which one we had. There were classic or FCD-two and I think we reported it in the paper. Some of them had a balloon cell and some did not. So type A and B.

Do you see a translational path, and I had the same question, do you see a translational path to treat people?

Dr. Bordey: The answer is yes. So the beauty of the HCN4 is that it’s not in the cortex, at least in adults or young adults. So it is a perfect target for gene therapy. It is expressed during development, it has not been studied very much, but it is there, and after birth it decreases tremendously. It’s expressed only in the thalamus, I think the amygdala and the cerebellum. So if targeting the cortex is a good gene therapy target, then can we use a drug? Presumably not because that would slow down the heart, the HCN4 is expressed in the heart and is important for their pacemaking activity, which I did not say, there are pacemaker channels, which also explain why they would maintain seizures, activity or cell firing.

Can we consider antisense oligonucleotide? Possibly. I think there is a lot of development by companies with small SIRNA that can be delivered intrathecally and they will last for about six months. HCN4 may be a good target. I think we need to know what is the function of HCN4 in the thalamus, because we would not want to block it there. It may be in the hippocampus a little bit. So it would be worth trying. I think it’s important actually to try whether a systemic SIRNA injection for covering the brain would work and have no side effects. So this is a big question. It’s just easier than gene therapy to move towards clinical application. So those are the two alternatives I can think of.

Have you tried blocking HCN4 after seizure onset?

Dr. Bordey: We did not. This is a very good question. We wanted to do it. We have the plasmid, we have not done it. We know you can block… When seizures are established, we know that we can block them and it is sufficient to… We can inject drug and it’s sufficient to block seizures. We have done that with Lena who showed that blocking translation after the onset of seizures, after they establish well decreased seizure frequency. So you can shrink the cell size and presumably remove ion channels since HCN4 is translation dependent. We have also shown that if you block a molecule called [philomena 00:29:54], and we published that last year. If you block the activity of philomena with a small molecule, once the seizures are established, so in mice that are, I think there were maybe two months of age, you do decrease seizure frequency. So I’m hopeful that doing that also in adult will work.

Do these genetic epilepsies respond well to vagal nerve stimulation or resective surgeries?

Dr. Bordey: Yes, resective surgeries. Yes. They respond well. I think they’re… I don’t know all the numbers on top of my head, but maybe 20 or 25% of the patients will go through surgeries, I may be wrong with the number, but roughly. Not everybody can go through surgeries, depending on the location and if they have too many of these malformations. Patients can have 1 to 50, which makes things complicated. And I think at least 50% of the patients will be seizure-free, I think. I think it depends on the clinical center, maybe 50 to 70, but very often seizures come back. So that’s a problem with the surgeries. The vagal nerve stimulation, I know it is used, and I do not remember the numbers, the test statistics in terms of efficacy. So I don’t know that.

Do you know the upstream genetic drivers of increased HCN4?

Dr. Bordey: We know it’s rapamycin sensitive, we saw it. So we use the plasmid Rheb, and Rheb is right upstream mTOR, and this is sufficient to increase HCN4. We have, I don’t think we published it, but we had a [inaudible 00:31:57] in TSC one [flocks 00:31:59] mice, so mute knockout mice. In TSC one, HCN4 four was increased as well. And since it’s increased in the patient, presumably it includes other genes since TSC one and Rheb, and then upstream, you have AKT and PI3K. But we have not looked at PI3K, we have not looked at P10 [inaudible 00:32:23] five, for example, to get to one complex, which I think is really important to do.

Do you know if it’s translational or is it the transcription? The up regulation?

Dr. Bordey: So it’s for ADP dependent, like I think Lena had shown, so it’s translation dependent. There is no increase of the MRNA, at least what we saw in our mice. And it’s not unexpected that you have cells with floating MRNA that is not translated, so we clearly promote a translation of that gene.

Is the HCN4 MRNA the same isoform as found in other parts of the brain or the heart? And if not, there may be potential for targeting?

Dr. Bordey: Yes, this is a good question. And I’ve asked myself that question and we have not, we have not checked. It is a possibility. So there are two different isoforms, a long and a short isoform, and I don’t remember which one the brain has, but the problem is we have not looked in the disease condition, which could even be a slightly different isoform. So I think that this is a very… This is something really important to do.

Do you know whether HCN4 is associated with other types of epilepsies?

Dr. Bordey: Yeah, actually I don’t remember the group that did it, but they found… I actually don’t remember right now, I should have checked. I know there is a mutation in HCN4 channel that has been reported, I think maybe last year, that is associated with seizures. I don’t remember if it’s a gain of function or loss of function. So there’s one study that reported that. There’s also genomic association studies showing that it actually could be associated with also a bipolar disorder since you control excitability. So I think this is quite… This is a gene of interest that people have not seen in the past because it’s a very discrete expression in the brain, so people thought very limited function and you have no clean blocker of HCN4. The blockers will touch all the HCN channels, so it has been hard to study by many. But now I think that our study highlights the importance of this channel in seizure, and perhaps, other disorders.


Postdoctoral Data Blitz on Translational Research in Epilepsy

CURE Epilepsy’s Frontiers in Research Seminar Series program is generously supported by the Nussenbaum-Vogelstein Family and aims to help educate and expose researchers, clinicians, and students to exciting epilepsy research and also provide opportunities for young investigators to interact with leaders in the field.

CURE Epilepsy conducted a virtual, postdoctoral data blitz, a series of short talks, to provide senior postdoctoral researchers an opportunity to share their work with other epilepsy researchers. The seminar focused on highly innovative, translational epilepsy research that can lead to a cure. The hour-long seminar highlighted work from the following three postdoctoral researchers, who each had ten minutes to present their work followed by five minutes for questions from the audience:

Maria Belen Perez-Ramirez, PhD
Stanford University
Focal Status Epilepticus and Gabapentin Effects on the Neocortex

Abstract: Status epilepticus (SE) is a severe neurological disorder with consequences ranging from death to increased risk for subsequent unprovoked seizures leading to epilepsy and cognitive abnormalities. An important unanswered question is whether a prolonged episode of focal status epilepticus (fSE) in the neocortex elicits long-lasting modifications leading to epileptogenesis. Dr. Perez-Ramirez is testing the hypothesis that alterations after a single episode of fSE result in structural and functional changes among neurons in neocortical circuits, potentially leading to epileptogenesis, and that treatment with an anti-synaptogenic agent, Gabapentin, may have prophylactic effects on structural, functional, and behavioral abnormalities following fSE.

Maria-Belen Perez-Ramirez is a biologist and obtained her PhD degree in Biomedical Sciences at the National University of Mexico. She is currently working as a PhD fellow in the neurology department at Stanford.

Lena Nguyen, PhD
Yale University
Targeting Translational Dysregulation in mTOR-related Epilepsy

Abstract: Hyperactivation of mTOR signaling due to mutations in mTOR pathway genes causes a spectrum of neurodevelopmental disorders (mTORopathies) associated with malformation of cortical development and intractable epilepsy. Dr. Nguyen’s research has led to the discovery that altered activity of the mTOR-dependent translational repressor 4E-BP1/2 contributes to neurodevelopmental defects and epilepsy in two prototypic mTORopathies, tuberous sclerosis complex (TSC) and focal cortical dysplasia type II (FCDII). The team is currently investigating novel and clinically relevant strategies (such as gene therapy) to target 4E-BP1/2 to treat epilepsy.

Lena Nguyen is a postdoctoral associate in Dr. Angelique Bordey’s lab at Yale University School of Medicine. Her research focuses on how alterations in intracellular signaling pathways contribute to epilepsy in neurodevelopmental disorders.

Rajeswari Banerji, PhD
University of Colorado
Enhancing Glucose Metabolism via Gluconeogenesis with PK11195 is Therapeutic in a Zebrafish Model of Dravet Syndrome

Abstract: Energy producing pathways are novel therapeutic targets for the treatment of neurodevelopmental disorders. Dr. Banerji’s research utilized a translatable zebrafish model of Dravet syndrome (scn1lab) which exhibits key characteristics of Dravet syndrome and shows metabolic deficits accompanied by downregulation of gluconeogenesis genes, pck1 and pck2. Treatment with a pck1 activator normalized dysregulated glucose levels, metabolic deficits, translocator protein expression and significantly decreased electrographic seizures in mutant larvae. Inhibition of pck1 in wild-type larvae mimicked metabolic and behavior defects observed in scn1lab mutants. Together, this suggests correcting dysregulated metabolic pathways can be therapeutic in neurodevelopmental disorders such as Dravet syndrome arising from ion channel dysfunction.

Rajeswari Banerji is a senior postdoctoral trainee in the Department of Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus. Her research involves understanding and developing novel metabolism-based therapies for neurological disorders, particularly epilepsy.

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Let’s start with a question that came in for Lena. They want to know, why did you focus on 4E-BP1 and not 4E-BP2?

Dr. Lena Nguyen: That’s a great question. There are three sub-types of 4E-BP, there’s 4E-BP1, 2, and 3. 1 and 2 is expressed in the brain, with 2 being the most highly expressed. At the time that we started this study, the tools for targeting 4E-BP1 was more readily available to us since this is the most common studied subtype in the cancer field. That is why we used it. Functionally, 4E-BP1 and 2 are very similar. The sequence hemology, I think is more than 60%. In our case, we over-expressed 4E-BP1 which would compensate for both the inhibited activity of 4E-BP1 and 2. I think that expressing constitutive active 4E-BP2 could potentially have the same effect.

Here’s a question for Belen. They want to know, how do you analyze your EEGs? And what criteria need to be met to establish that there is seizure activity in your models?

Dr. Maria Belen Perez-Ramirez: Thanks for the interest. To analyze the EEG, we use Power Analysis. Please remember that we induce the seizure activity at the beginning but we haven’t still studied later EEG activity. But then to establish that there is seizure activity, there needs to be interictal electrographic discharges, and then I also expect to have behavioral contralateral jerkings from the animal. When the animal has these contralateral behavioral seizures together with the interictal seizures, then that’s when I start counting the time, the two hour period of seizure activity. Then something we notice is that after their interictal spikes, they can develop to ictal activity. Then we just let the seizure activity for two hours and then we stop the seizure. But there should be both behavioral and electrographic interictal spikes.

I’m going to move onto Raji. There’s a question here, and they want to know please tell us more about PK11195? Do we know anything about the structure? Does it get into the brain? Has it been tested in other models of epilepsy?

Dr. Rajeswari Banerji: That’s a great question. Yes, we know a lot about this drug. This had been discovered in 1977, so a lot of research has been done and so this drug is a synthetic GSP11, so it binds to the mitochondrial ligand that is the translocator protein. It is also known as the peripheral benzodiazepine receptor, so it does have a lot of information on how it mines and how it producing neuro [inaudible 00:41:38], so there’s a lot of research going on. As I mentioned in the talk, there are a few papers which have shown an anti-epileptic property of this drug. We are really excited to try this in our new model, and also try it in mice models.

Raji, do you know if the efficacy of this drug, this anti-seizure activity, is that dependent on its improvement of the metabolic activity or are they two separate things?

Dr. Banerji: No, what we think, certainly after doing three years of research on our zebrafish model, that a metabolic deficit is required for this drug to work. We think this drug may work as a censor, actually, because we don’t think it always improves. It may reduce the glucose levels, also. A better term will be normalize, so it senses any kind of defects, metabolic defects, and then corrects it. We still don’t know how it corrects a seizure phenotype, and that’s something which we have to understand using other models maybe. But that’s a good question also. Thank you.

Lena, back to you. The high variability in your seizure rescue finding is really interesting. Do you have any clues on why many saw complete reduction in seizures, while others looked similar to the control group? Did you find that seizure frequency correlated with any of your other measures?

Dr. Nguyen: That’s a great question. Several things could contribute to this, one of which is a technical consideration for in-utero electroporation. We have been pretty good at getting consistent with the area that we target and the size of the targeted area, but when electroporating multiple plasmid as we did, not all cells are going to end up with all of the four plasmid. The majority will, but not all. These differences could contribute to the large variability. In addition to that, differences with combination efficiencies could lead to different levels of 4E-BP expression, which could also contribute to this. In our case, cell size is a good readout of whether 4E-BP activity worked the way we had intended it to, and so we have correlated cell size with seizure activity and we found a very strong correlation where those with no or low seizure activity following 4E-BP expression had smaller cell size, and those with higher seizure frequency had larger cells, suggesting that 4E-BP might be insufficient. It is interesting that we still see such a strong effect despite this variability, and we might actually be underestimating the effects of 4E-BP on seizures.

Lena, have you looked at or has anyone else looked at different models of cortical malformations to know is there 4E-BP1, is it also hyperphosphorylated in other models? Is that known?

Dr. Nguyen: Other groups have shown that [inaudible 00:45:23] is increased in human mTORopathies, like TFC and FCD2, and increased phosphorylation is expected as well in other mouse models.

Next question is back to Belen. What would your next steps be to translate your findings to people who have experienced status? Have you tried Gabapentin animal models of epilepsy?

Dr. Perez-Ramirez: Thanks. Yeah, that’s a complex question, the first one. Yes. A bit, first, I think we need to find what will be the best regime of Gabapentin, or Gabapentin treatment. Meaning the concentration and timing, because also there is a time window as far as we know where this is synaptogenic activity. We still don’t know in this model, what is this time window and the latency? That’s also another issue we need to address. Also, it will be important that once we know the most efficient Gabapentin treatment regime, then how this translates to human. Gabapentin is already an FDA-approved drug, so we just need to understand how the doses even after status epilepticus can work in humans. Then as I was saying, the window will be very important as also Gabapentin, it’s as far as we know, the efficacy happens also with newly formed synapses. That’s why it is important to know the latency, or the synaptogenic period.

About the second question, was Gabapentin trialed in other models? In the lab here, it was already tested on the undercut model of epilepsy, and they found similar results with decreases in alpha 2 delta 1, and decreases in synaptic activity, and also decreases in synaptic connectivity. Then there is the group from Rossi, and they tried Gabapentin in the pilocarpine model of temporal epilepsy, and I think they found also that Gabapentin was able to decrease astrocytic activity. In the latest article, they test this with animals that were treated with PILO and then treated with Gabapentin, and then given a second dose of PILO, and they found that there was decreased mortality, decreased racine scale achieved, and decreased animals that get to actual status epilepticus on that second dose. And then, there is the group of [inaudible 00:49:01] that they tried Gabapentin on a model of cortical malformation, and they also found decreased epileptiform activity with Gabapentin and I think also decreased expression of thrombospondins. It’s been tried in different models.

You mentioned about timing, so have you looked at, I think you’re doing it at three hours after status, the Gabapentin treatment you initiated? Have you tried other time points, like starting it later?

Dr. Perez-Ramirez: That sort of thing, we are interested in looking at.

Thank you. I had another question for Lena, just a technical question. In the samples from the humans where you showed the 4E-BP1 hyper-phosphorylation, what did you use as controls for those?

Dr. Nguyen: Because they were human samples, [inaudible 00:50:20] come across, we have not been able to get control human tissue samples. But there have been other studies where 4E-BP at this level is not found in the control tissue. We also looked at surrounding cells just to see. It’s not the best control, but those cells did not have increase. It was very specific to the dysmorphic enlarged neurons, the increase.