ARTICLE IN BRIEF
Investigators reported that mice in which toll-like receptor 3 had been knocked out were found to experience about 80 percent fewer spontaneous seizures on average than wild-type mice following treatment with the anticholinergic drug, pilocarpine, a standard means of inducing status epilepticus-provoked epilepsy.
An important new piece of the puzzle of inflammatory mediation in epileptogenesis has been identified by a team of Israeli researchers.
In a platform presentation at the AAN Annual Meeting in Philadelphia in April, the group presented the first evidence that toll-like receptor 3 (TLR3), a molecular mediator of innate immunity, plays a key role in setting off epileptic seizures in response to brain insults.
Mice in which TLR3 had been knocked out were found to experience about 80 percent fewer spontaneous seizures on average than wild-type mice following treatment with the anticholinergic drug, pilocarpine, a standard means of inducing status epilepticus–provoked epilepsy.
“We looked at TLR3 because it's an intracellular receptor expressed widely in the CNS that can be activated by viruses, stroke, trauma — all the sorts of injuries associated with epileptogenesis,” said the first author of the paper, Felix Benninger, MD, PhD, a last-year resident in neurology at the Rabin Medical Center in Petah Tikva, Israel. “It's a very specific inflammatory reaction to a broad range of injuries.”
While wild-type mice had an average of 2.5 stage III or IV seizures per day, as measured by implanted EEG as well as video monitoring, TLR3 knockout mice averaged just 0.3 per day (p=0.0076).
The wild-type mice experienced 20 milder stage I or II seizures per day on average, compared with just five per day for the TLR3 knockouts (p=0.0004). In all, 500 mice were used in the study, half of them knockouts and half wild-type.
“This is the first extensive study demonstrating the role of TLR3 in generating seizures,” said Annamaria Vezzani, PhD, a neuroscientist at the Mario Negri Institute for Pharmacological Research in Milan, Italy, where she has been a pioneer in exploring the role of inflammation in epileptogenesis. “We published a similar experiment in mice lacking TLR4 and found very similar results. So there might be a kind of synergy between the activation of TLR3 and TLR4 in epileptogenesis, but we don't know yet.”
Recognition that toll-like receptors play a role in epilepsy is very recent, Dr. Vezzani said, but should not be all that surprising.
“It's been well known that infections of the brain are a major risk factor for developing epilepsy in humans,” she said. “The toll-like receptors are among the key means the body has for recognizing bacterial or viral products. They also recognize molecules released by brain cells following epileptogenic injuries or during seizures. They have been shown to increase the risk of seizures, and seizures have in turn been shown to activate toll-like receptors. So it becomes a vicious, pathologic, self-sustaining circle.”
Another leading researcher investigating the role of inflammation in epileptogenesis cautioned that the study is but a first, if essential, step in understanding how TLR3 fits into the puzzle. “The knockouts are a first step,” said Jacqueline French, MD, FAAN, a professor of neurology at New York University (NYU) Medical Center and a member of the Neurology Today editorial advisory board. “It's another indication that targeting inflammation is potentially the right way to go. Hopefully it will spur more research into therapies that can interrupt the epileptogenesis process.”
Dr. French, who is director of the clinical trials consortium at NYU's Comprehensive Epilepsy Center, co-chaired a symposium at last year's AAN meeting on inflammation and epilepsy.
“This is an evolving field, which has come very much into sharp focus in just the last two years,” she said. “The idea arose from observations in both animal models and in human beings that signs of ongoing inflammation are seen around a seizure focus. When you take tissue from a patient undergoing epilepsy surgery and look at it under the microscope, you see increased astrocytes, microglia and an increase in inflammatory intermediators, such as interleukin 1 beta, high mobility group box1, tumor necrosis factor and interleukin 6. It's an active process that kicks up every time there is another seizure.”
The toll gene was first identified in 1985 by a German researcher, Christiane Nüsslein-Volhard, who first observed how a mutated version it caused drosophila larva to have a bizarrely underdeveloped underside, Dr. French explained. The protein encoded by the gene was later found to be similar to a family of other proteins, dubbed toll-like receptors, all of which function as a pivotal part of the innate immune system.
The hope of finding an immune-moderating treatment for epilepsy is that it could actually prevent the disorder from developing following a brain injury or infection, said Hal Blumenfeld, MD, PhD, professor of neurology, neurobiology and neurosurgery at the Yale University School of Medicine.
“We do not yet have any treatments that prevent the disease,” Dr. Blumenfeld said. “It would be reasonable to think about clinical trials to prevent inflammation in populations known to be at risk for developing epilepsy, for example following head trauma.”
But such translational trials could prove challenging, said Dr. French. “Most of the things that cause epilepsy, such as head trauma or stroke, lead to epilepsy only in about one in ten people,” she said. “So whatever the intervention is, you're going to have to give it to 10 people to prevent epilepsy in one. And if the drug was only 30 percent effective, you'd have to give it to more than 30 people to prevent one case of epilepsy. So it would have to be something we know is pretty safe.”
A few investigational agents are now being explored that target both TLR3 and 4, researchers said. But they are a long way from becoming available, Dr. Benninger said.
Dr. Vezzani and others have also been exploring the role of an investigational agent known as VX-765, which has been found to block inflammation and epileptogenesis in mice. A human clinical trial of VX-765 carried out by another group, however, was stopped early by the drug company funding it for financial reasons, said Dr. French.
“We still don't have a smoking gun to say we know for a fact that we can intervene in the inflammatory process and prevent epilepsy,” Dr. French said. “If something does work, the beauty is that all the other antiepileptic drugs we have just target the symptoms. An anti-inflammatory drug could target the underlying disease.”
EXPERTS: ON TOLL-LIKE RECEPTORS READY FOR EPILEPSY
•. AAN Annual Meeting Abstract: The role of toll-like receptor 3 in epileptogenesis: http://bit.ly/1nN6c8W
•. Hansson GK, Edfeldt K. Toll to be paid at the gateway to the vessel wall. Arterioscler Thromb Vasc Biol. 2005; 25:(6): 1085–1087.
•. Iori V, Maroso M, Rizzi M, et al. A receptor for advanced glycation endproducts is upregulated in temporal lobe epilepsy and contributes to experimental seizures. Neurobiol Dis. 2013; 58:102–114.
•. Maroso M, Balosso S, Ravizza T, et al. Interleukin-1 biosynthesis inhibition reduces acute seizures and drug resistant chronic epileptic activity in mice. Neurotherapeutics. 2011; 8:(2): 304–315.
•. Maroso M, Balosso S, Ravizza T, et al. Toll-like receptor 4 and high-mobility group box-1 are involved in ictogenesis and can be targeted to reduce seizures. Nat Med. 2010; 16:(4): 413–419.
•. Youn HS, Lee JY, Fitzgerald KA, et al. Specific inhibition of MyD88-independent signaling pathways of TLR3 and TLR4 by resveratrol: molecular targets are TBK1 and RIP1 in TRIF complex. J Immunol. 2005; 175:(5): 3339–3346.