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.