Researchers reveal new cause of epilepsy

A team of researchers from Sanford-Burnham and SUNY Downstate Medical Center has found that deficiencies in hyaluronan, also known as hyaluronic acid or HA, can lead to spontaneous epileptic seizures. HA is a polysaccharide molecule widely distributed throughout connective, epithelial, and neural tissues, including the brain’s extracellular space (ECS). Their findings, published on April 30 in The Journal of Neuroscience, equip scientists with key information that may lead to new therapeutic approaches to epilepsy.

The multicenter study used mice to provide the first evidence of a physiological role for HA in the maintenance of brain ECS volume. It also suggests a potential role in human epilepsy for HA and genes that are involved in hyaluraonan synthesis and degradation.

While epilepsy is one of the most common neurological disorders—affecting approximately 1 percent of the population worldwide—it is one of the least understood. It is characterized by recurrent spontaneous seizures caused by the abnormal firing of neurons. Although epilepsy treatment is available and effective for about 70 percent of cases, a substantial number of patients could benefit from a new therapeutic approach.

“Hyaluronan is widely known as a key structural component of cartilage and important for maintaining healthy cartilage. Curiously, it has been recognized that the adult brain also contains a lot of hyaluronan, but little is known about what hyaluronan does in the brain,” said Yu Yamaguchi, M.D., Ph.D., professor in our Human Genetics Program.

“This is the first study that demonstrates the important role of this unique molecule for normal functioning of the brain, and that its deficiency may be a cause of epileptic disorders. A better understanding of how hyaluronan regulates brain function could lead to new treatment approaches for epilepsy,” Yamaguchi added.

The extracellular matrix of the brain has a unique molecular composition. Earlier studies focused on the role of matrix molecules in cell adhesion and axon pathfinding during neural development. In recent years, increasing attention has been focused on the roles of these molecules in the regulation of physiological functions in the adult brain.

In this study, the investigators examined the role of HA using mutant mice deficient in each of the three hyaluronan synthase genes (Has1, Has2, Has3).

“We showed that Has-mutant mice develop spontaneous epileptic seizures, indicating that HA is functionally involved in the regulation of neuronal excitability. Our study revealed that deficiency of HA results in a reduction in the volume of the brain’s ECS, leading to spontaneous epileptiform activity in hippocampal CA1 pyramidal neurons,” said Sabina Hrabetova, M.D., Ph.D., associate professor in the Department of Cell Biology at SUNY.

“We believe that this study not only addresses one of the longstanding questions concerning the in-vivo role of matrix molecules in the brain, but also has broad appeal to epilepsy research in general,” said Katherine Perkins, Ph.D., associate professor in the Department of Physiology and Pharmacology at SUNY.

“More specifically, it should stimulate researchers in the epilepsy field because our study reveals a novel, non-synaptic mechanism of epileptogenesis. The fact that our research can lead to new anti-epileptic therapies based on the preservation of hyaluronan adds further significance for the broader biomedical community and the public,” the authors added.

Commonly available blood-pressure drug prevents epilepsy after brain injury

Between 10 and 20 percent of all cases of epilepsy result from severe head injury, but a new drug promises to prevent post-traumatic seizures and may forestall further brain damage caused by seizures in those who already have epilepsy.

A team of researchers from UC Berkeley, Ben-Gurion University in Israel and Charité-University Medicine in Germany reports in the current issue of the journal Annals of Neurology that a commonly used hypertension drug prevents a majority of cases of post-traumatic epilepsy in a rodent model of the disease. If independent experiments now underway in rats confirm this finding, human clinical trials could start within a few years.

“This is the first-ever approach in which epilepsy development is stopped, as opposed to common drugs that try to prevent seizures once epilepsy develops,” said coauthor Daniela Kaufer, UC Berkeley associate professor of integrative biology and a member of the Helen Wills Neuroscience Institute. “Those drugs have a very limited success and many side effects, so we are excited about the new approach.”

The team, led by Kaufer; neurosurgeon Alon Friedman, associate professor of physiology and neurobiology at the Ben-Gurion University of the Negev; and Uwe Heinemann of the Charite, provides the first explanation for how brain injury caused by a blow to the head, stroke or infection leads to epilepsy. Based on 10 years of collaborative research, their findings point a finger at the blood-brain barrier – the tight wall of cells lining the veins and arteries in the brain that is breached after trauma.

“This study for the first time offers a new mechanism and an existing, FDA-approved drug to potentially prevent epilepsy in patients after brain injuries or after they develop an abnormal blood-brain barrier,” Friedman said.

The drug, losartan (Cozaar®), prevented seizures in 60 percent of the rats tested, when normally 100 percent of the rats develop seizures after injury. In the 40 percent of rats that did develop seizures, they averaged about one quarter the number of seizures typical for untreated rats. Another experiment showed that administration of losartan for three weeks at the time of injury was enough to prevent most cases of epilepsy in normal lab rats in the following months.

“This is a very exciting result, telling us that the drug worked to prevent the development of epilepsy and not by suppressing the symptoms,” Kaufer said.

Breakdown of the blood-brain barrier

Kaufer and Friedman have been collaboratively investigating the effects of trauma on the brain since Kaufer was a graduate student in Israel 20 years ago. Throughout a postdoctoral position at Stanford University and after joining the UC Berkeley faculty in 2005, she maintained her interest in the blood-brain barrier, which normally protects the brain from potentially damaging chemicals or bacteria in the blood and prevents brain chemicals from leaking into the blood stream.

She and Friedman showed earlier that breaking down the barrier causes inflammation and leads to the development of epilepsy. They pinned the effect to a single protein called albumin, the most common protein in blood serum.

In 2009, they showed that albumin affects astrocytes, the brain’s support cells, by binding to the TGF-β (transforming growth factor-beta) receptor. This initiates a cascade of steps that lead to localized inflammation, which appears to permanently damage the brain’s wiring, leading to the electrical misfiring characteristic of epilepsy. The current paper conclusively demonstrates that blocking the TGF-beta receptor with losartan stops that cascade and prevents the disorder.

Drug’s side effect proves crucial

Coauthor Guy Bar-Klein, a doctoral student at Ben-Gurion University, searched a long list of drugs before discovering losartan, which is approved to treat high blood pressure because it blocks the angiotensin receptor 1, but which incidentally also blocks TGF-β. It worked in the rats when delivered in their drinking water, which means that it somehow gets into the brain through the blood-brain barrier. The experiments suggest that the drug is unable to cross an intact blood-brain barrier, but reaches the brain through a breached barrier when it is most needed, Kaufer said.

Friedman developed a protocol to use MRI to check whether the blood brain barrier has been breached, allowing doctors to give losartan as a preventive treatment, if necessary, after trauma. Kaufer said that the barrier may remain open for only a few weeks after injury, so the drug would not have to be given very long to prevent damage.

“Right now, if someone comes to the emergency room with traumatic brain injury, they have a 10 to 50 percent chance of developing epilepsy, and epilepsy from brain injuries tends to be unresponsive to drugs in many patients.” she said. “I’m very hopeful that our research can spare these patients the added trauma of epilepsy.”

A single switch dictates severity of epileptic seizures

A switch in the brain of people with epilepsy dictates whether their seizures will be relatively mild or lead to a dangerous and debilitating loss of consciousness, Yale researchers have found.

The study published April 11 in the journal Neurology showed that there was no gradation of impairment during seizures — subjects were either alert or totally unaware of their surroundings.

The existence of an “all or none” switch for consciousness surprised researchers, who expected to find different levels of awareness among those who experience focal seizures, or those localized to particular brain areas.

“During seizures patients may report a funny, fearful feeling, tingling in their arm or some quirk in their vision but are able to answer all our questions,” said Dr. Hal Blumenfeld, professor of neurology, neurobiology, and neurosurgery, and senior author of the study. “At other times — boom — all of a sudden they are in a daze, unable to respond to their environment.”

Blumenfeld said previous studies have shown that this switch rests in areas of the brain stem that play a role in waking and in paying attention to your surroundings. The findings suggest that existing drugs that treat narcolepsy or therapies like deep brain stimulation might help patients with intractable epilepsy.

“Our goal is to prevent seizures, but in a fifth to a quarter of people have seizures no matter what we do,” Blumenfeld said. “For them, therapies that would prevent loss of consciousness would greatly improve quality of life.”

Sleep-dependent memory consolidation and accelerated forgetting

Accelerated long-term forgetting (ALF) is a form of memory impairment in which learning and initial retention of information appear normal but subsequent forgetting is excessively rapid. ALF is most commonly associated with epilepsy and, in particular, a form of late-onset epilepsy called transient epileptic amnesia (TEA). ALF provides a novel opportunity to investigate post-encoding memory processes, such as consolidation. Sleep is implicated in the consolidation of memory in healthy people and a deficit in sleep-dependent memory consolidation has been proposed as an explanation for ALF. If this proposal were correct, then sleep would not benefit memory retention in people with ALF as much as in healthy people, and ALF might only be apparent when the retention interval contains sleep. To test this theory, we compared performance on a sleep-sensitive memory task over a night of sleep and a day of wakefulness. We found, contrary to the hypothesis, that sleep benefits memory retention in TEA patients with ALF and that this benefit is no smaller in magnitude than that seen in healthy controls. Indeed, the patients performed significantly more poorly than the controls only in the wake condition and not the sleep condition. Patients were matched to controls on learning rate, initial retention, and the effect of time of day on cognitive performance. These results indicate that ALF is not caused by a disruption of sleep-dependent memory consolidation. Instead, ALF may be due to an encoding abnormality that goes undetected on behavioural assessments of learning, or by a deficit in memory consolidation processes that are not sleep-dependent.

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(Image: Courtney Icenhour)

Study in Fruit Flies Shows that Epilepsy Drug Target May Have Implications for Brain Disorder Sleep Disruption

A new study in a mutant fruitfly called sleepless (sss) confirmed that the enzyme GABA transaminase, which is the target of some epilepsy drugs, contributes to sleep loss. The findings, published online in Molecular Psychiatry, were led by Amita Sehgal, PhD, head of the Chronobiology Program at the University of Pennsylvania’s Perelman School of Medicine. The findings shed light on mechanisms that may be shared between sleep disruption and some neurological disorders. A better understanding of this connection could enable treatments that target both types of symptoms and perhaps provide better therapeutic efficacy.

“Epilepsy is essentially an increase-in-firing disorder of the brain and maybe a decrease in activity of the neurotransmitter GABA, too,” says Sehgal, who is also a professor of Neuroscience and an investigator with the Howard Hughes Medical Institute (HHMI). “This connects our work to drugs that inhibit GABA transaminase. Changes in GABA transaminase activity are implicated in epilepsy and some other psychiatric disorders, which may account for some of the associated sleep problems.”

The team looked at the proteomics of the sss mutant brain – a large-scale study of the structure and function of related proteins — and found that GABA transaminase is increased in the sss brain compared to controls. This enzyme breaks down GABA, so GABA is decreased in the sss brain. Because GABA promotes sleep, there is a decrease in sleep in the sss mutant fly, as the name implies.  

The relationship between the SSS protein and GABA is not fully understood. The SSS protein controls neural activity, and its absence results in increased neural firing, which likely uses up a lot of energy, says Sehgal. GABA transaminase works in the mitochondria, the energy-production organelle in the glial cells of the brain, which provide fuel for neurons. The large energy demand created by the increased neural firing in sss brains probably alters mitochondrial metabolism, including GABA transaminase function in glia.

In the sss mutant fly, there is a stream of connections that leads to its signature loss of sleep: The sss mutant has increased neuron firing caused by downregulation of a potassium channel protein called Shaker. Recently, the Sehgal lab showed that SSS also affects activity of acetylcholine receptors. Both of these actions may directly cause an inability to sleep. In addition, increased energy demands on glia, which increase GABA transaminase and decrease GABA, may further contribute to sleep loss. On the other hand, if GABA is increased, then sleep is increased, as in flies that lack GABA transaminase.

Researchers reveal the dual role of brain glycogen

In 2007, in an article published in Nature Neuroscience, scientists at the Institute for Research in Biomedicine (IRB Barcelona) headed by Joan Guinovart, an authority on glycogen metabolism, suggested that in Lafora Disease (LD), a rare and fatal neurodegenerative condition that affects adolescents, neurons die as a result of the accumulation of glycogen—chains of glucose. They went on to propose that this accumulation is the root cause of this disease.

The breakthrough of this paper was two-sided: first, the researchers established a possible cause of LD and therefore were able to point to a plausible therapeutic target, and second, they discovered that neurons have the capacity to store glycogen—an observation that had never been made—and that this accumulation was toxic.

Other reports defended a different theory and upheld that the glycogen deposits were not cause by the neurodegeneration but were a consequence of another, more important, cell imbalance, such as a down deregulation of autophagy—the cell recycling and cleaning programme. In several articles, Guinovart’s “Metabolic engineering and diabetes therapy” group has recently brought to light evidence of the toxicity of glycogen deposits for LD patients, and has now provided irrefutable data.

In an article published at the beginning of February in Human Molecular Genetics, with the research associate Jordi Duran as first author, the scientists show that in LD the accumulation of glycogen directly causes neuronal death and triggers cell imbalances such a decrease in autophagy and synaptic failure. All these alterations lead to the symptoms of LD, such as epilepsy.

Glycogen, a Trojan horse for neurons?

There was still a greater mystery to be solved. Was glycogen synthase truly a Trojan horse for neurons, as apparently established in the article in Nature Neuroscience? That is to say, was the accumulation of glycogen always fatal for cells, thus explaining why their glycogen synthesis machinery is silenced? The inevitable question was then why these cells had such machinery.

In another paper published in Journal of Cerebral Blood Flow & Metabolism, part of the Nature Group, the researchers provided the first evidence that neurons constantly store glycogen but in a different way: accumulating small amounts and using it as quickly as it becomes available. In this regard, the scientists set up new, more sensitive, analytical techniques to confirm that the machinery responsible for glycogen synthesis and degradation existed. In summary, they showed that, in small amounts, glycogen is beneficial for neurons.

“For example, while the liver accumulates glycogen in large amounts and releases it slowly to maintain blood sugar levels, above all when we sleep, neurons synthesize and degrade small amounts of this polysaccharide continuously. They do not use it as an energy store but as a rapid and small, but constant, source of energy,” explains Guinovart, also senior professor at the University of Barcelona (UB).

To observe the action of glycogen, the scientists forced cultured mouse neurons to survive under oxygen depletion. They demonstrated that the first cells to die were those in which the capacity to synthesise glycogen had been removed. The same experiments were performed in collaboration with Marco Milán’s “Development and growth control” group in the in vivo model of the fruit fly Drosophila melanogaster. These tests led to the same conclusions.

The researchers postulated that glycogen is a lifeguard under oxygen depletion, a condition that leads the brains to shut down and that often occurs at birth and in cerebral infarctions in adults, which leads to severe consequences, such a cerebral paralysis.

“It is the first function of glycogen that we have discovered in neurons, but we still have to identify its function in normal conditions and establish how the mechanism works,” says Jordi Duran. Postdoctoral researcher Isabel Saez is the first author of the article out today, which involved the collaboration of ICREA Research Professor Marco Milán’s lab.

The beneficial and toxic roles of brain glycogen are currently the focus of main research lines conducted by Joan Guinovart’s lab.

Seizing Control of Brain Seizures

A few years after serving in the Israeli army during the first Gulf War, Daniela Kaufer made a startling discovery about the effect of psychological stress on the brain. As a graduate student at the Hebrew University she showed that the kind of extreme stress experienced in combat can break down the physiological barriers that normally protect the brain.

She could not have known it then, but the finding would eventually lead her to uncover a key change in brain chemistry that triggers epileptic seizures. The Bakar Fellows Program is now helping her refine a strategy to block the threat and protect the brain from damage caused by physical trauma and other insults.

A physiological line of defense normally prevents circulating blood from entering the brain. Known as the blood-brain barrier, the tightly controlled system buffers the brain from exposure to bacteria and other blood-borne invaders. Kaufer’s research has revealed how brain trauma can disrupt brain function once the barrier is breached.

In lab research as a postdoc at Stanford in 2002, Kaufer and her Israeli colleague Alon Friedman examined what happens in the brain when the barrier is compromised. They found that seizures were likely if – and only if – the brain came in contact with blood that had been circulating in the body.

They showed that a very common protein in blood called albumin accelerates signaling between neurons to abnormal levels. Neurons become overexcited and can cause seizures.

“We were surprised, even a little disappointed, that it was such a common component of the blood  – nothing exotic at all  – that led to epilepsy,” recalls Kaufer, associate professor of integrative biology.

She and Friedman went to on to show that albumin interacts with a ubiquitous cell protein called TGF-Beta receptor to cause the damage.

In the healthy brain, TGF-Beta signaling affects activity of star-shaped sister cells of neurons called astrocytes, which normally limit neuron-to-neuron firing signals across the synapse. But when albumin stimulates TGF-Beta receptors, astrocytes lose some of their control. Neuron signaling spikes dangerously, and promotes the development of epileptic seizures.

“Researchers knew that following traumatic brain injury the risk of epilepsy was great, but they didn’t know why,” Kaufer says.

As luck would have it, a prescription drug for hypertension blocks TGF-Beta signaling.  With support from the Bakar Fellows program, Kaufer is now carrying out research to confirm that blocking abnormal TGF-Beta activity can prevent epilepsy from a range of insults.

She expects that her and Friedman’s lab research, coupled with clinical studies, will demonstrate the drug’s ability to protect the brain and move it into use in emergency medicine to prevent victims of brain trauma from becoming epileptic.

Kaufer and Friedman’s research is suggesting too that a number of assaults besides physical trauma – from brain infections to stroke – can also weaken the blood-brain barrier, and lead to the development of epilepsy through TGF-beta signaling. Emergency medicine physicians need only determine if the barrier has been breached to know if a patient is at risk for seizures.

Fortunately, the condition of the blood-brain barrier can be assessed using a safe and  straightforward FDA-approved MRI protocol, so screening for epilepsy risk is within reach, says Kaufer.

“Right now, if someone comes to the emergency room with traumatic brain injury, they have a 10 to 50 percent chance of developing epilepsy. But you don’t know which ones, nor do you have a way of preventing it. And epilepsy from brain injuries is the type most unresponsive to drugs.

“I’m very hopeful and that our research can spare these patients the added trauma of epilepsy.”

Tiny Proteins Have Outsized Influence On Nerve Health

Mutations in small proteins that help convey electrical signals throughout the body may have a surprisingly large effect on health, according to results of a new Johns Hopkins study published in Proceedings of the National Academy of Sciences in December using spider, scorpion and sea anemone venom. 

The tiny conduits carrying those electrical signals are sodium channels that are vital to our well-being—they trigger action potentials, or spurts of electrical energy that course from body to brain to deliver messages that invoke feelings like pain or temperature sensitivity. When such channels go awry, they contribute to a slew of diseases, one of which is epilepsy.

In the new research, Frank Bosmans, Ph.D., an assistant professor of physiology at the Johns Hopkins University School of Medicine, has found that auxiliary “helper” proteins that interact with sodium channels also play a crucial role. And that, he says, could affect drug development for epilepsy, neurological diseases, muscular disorders and pain syndromes.  

“Nobody had thought these tiny molecules that don’t even form the main sodium channel were capable of changing the response of the channel to certain compounds,” Bosmans says. “But in what we consider a new concept, these auxiliary subunits can be considered as drug targets.”

Over the past few decades, there have been hints that these auxiliary proteins were influencing sodium channels, but few analyzed the problem very closely. John Gilchrist, a graduate student in Bosmans’ lab, began evaluating each of the four proteins, one at a time.

Gilchrist engineered frog eggs that made sodium channels and exposed them to the toxins released by tarantulas, scorpions, wasps and sea anemones, an extension of Bosmans’ earlier doctoral research studying the effect of animal venoms on sodium channels. He found that one auxiliary protein in particular, beta4, altered the whole sodium channel system. When exposed to tarantula venom, for instance, tissue in the presence of beta4 showed decreased sensitivity in the sodium channels, meaning that the protein changed the way the nerve fired. This denotes that if a human got bit by a tarantula in a region where beta4 was active, the whole experience might be just a little less painful, says Bosmans.

To figure out what was going on in the altered channels, Bosmans needed to know what the protein looked like, he says. He contacted Filip Van Petegem, a crystallographer at the University of British Columbia in Vancouver, Canada. Van Petegem was able to map the 3-D structure of beta4 down to 1.7 angstroms, the highest possible resolution. Crystal structure in hand, Bosmans could now mutate beta4 and watch what happened. 

Purely by chance, Van Petegem had already started that mutation process. To diagram the crystal, Van Petegem had been forced to substitute one protein for another due to quirks in the test system. Bosmans found that the tiny mutation thwarted beta4’s interaction with the sodium channel system.

That finding promptly overturned conventional wisdom into how these proteins behave, Bosmans says. 

Back in 1998, Bosmans says, physicians determined that a mutation in the beta1 protein seemed to be triggering a case of epilepsy. Epilepsy has hundreds of causes. It was known at the time that a chemical bridge within the sodium channel held the beta proteins together. If that bridge, known as a disulfide bond, is broken, the proteins fall apart. The physicians theorized that the mutation they found must have destroyed the bridge along with their accompanying proteins. That broken bridge theory has remained dominant ever since.

But when Bosmans introduced that same mutation in beta4, the structure stayed intact. The changes he saw were much more subtle. The position of the protein Van Petegem had mutated changed slightly so that it was farther away from the channel. And only when that mutated crystal was exposed to a toxin did beta4 lose its ability to communicate with the sodium channel.

Bosmans says that even with evidence of the auxiliary proteins’ importance mounting, such as in the epilepsy study, drug developers have continued to ignore the proteins rather than treatment opportunities. Most efforts to develop new drugs to treat epilepsy still focus exclusively on modifying the sodium channels, which don’t need the beta proteins to operate. But Bosmans believes this is only part of the story.

His new finding suggests that such an approach is shortsighted, because mutations in these beta proteins may very well be causing the disease at hand. Drugs that target the beta proteins have the potential to deliver a much more focused treatment, he says.

"That’s one of the new concepts that we’re trying to launch—keep an eye on these little guy proteins, because they are important. If they have a mutation in them, they can cause a disease,” Bosmans says. 

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