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Discovering new mechanisms and therapies for epilepsies in children

Epilepsy is one of the most common neurological disorders worldwide, in which disturbances of the normal nerve cell activity in the brain leads to spontaneous seizures. While some forms of epilepsy are triggered by another disease such as brain infection, stroke or cancer, or can arise as the result of an external physical trauma during accidents or falls, the cause of most forms of epilepsy that appear in early childhood is not yet clearly understood. Recent studies have confirmed a strong genetic influence, but there are still many forms for which no adequate treatment is available.

“We are currently looking into the genetic origins of Dravet syndrome and related epilepsies. Children with these diseases have seizures that do not respond to the available medication and have severe developmental delays,” says Dr Alexander Crawford, Head of the Chemical Biology group at the Luxembourg Centre for Systems Biomedicine (LCSB) of the University of Luxembourg. To find the genetic origin of these diseases, Crawford collaborates with clinicians and genetics researchers at the LCSB and other research institutions who perform genome sequencing of affected children and their healthy parents. They are looking for genetic differences between the parents and the child which likely point to new, so-called de novo, mutations in specific genes that could cause these diseases.

Once the team has identified a potential epilepsy-causing mutation, they need to confirm this finding to be sure that this gene is indeed triggering the seizures. For that, the researchers need to rely on animal experimentation. The scientists chose the zebrafish (Danio rerio) which shows a characteristic swimming pattern during epileptic seizures and has several additional advantages that make it a suitable epilepsy model. The zebrafish is a small tropical freshwater fish originating from the southeastern region of the Himalayas. It is an important and widely-used vertebrate model organism in scientific research, particularly in the fields of developmental biology, functional genomics, disease modeling and drug discovery. Experiments are performed in zebrafish embryos and larvae, which are microscopically small and optically translucent so that one can follow their development under the microscope within the course of hours.

“Over 70% of the human genes are conserved in zebrafish, which enables us to study their role in the brain,” explains Crawford. With his team, he introduces in the fish either the same gene mutation that potentially causes epilepsy found in the patients or small specific blocking sequences that inactivate the function of the gene. To this end, they inject modified genetic material into fertilized eggs, which will change the activity of the gene of interest. For a period between one and five days, the researchers closely monitor the swimming behaviour of the larvae using automated tracking devices as well as recording the brain activity using electroencephalography techniques. If the genetically modified larvae show different swimming behaviour and increased brain activity in comparison with the untreated control larvae, this suggests that the artificially introduced patient mutation indeed causes epilepsy in the zebrafish model.

Once such a disease-causing gene mutation is found, the researchers can look deeper into the mechanism by which the mutation causes a disruption in normal nerve cell activity and look for drugs that could treat the seizures. To this end, Crawford and his team screen the genetically modified fish larvae with a library of drug-like molecules including both approved drugs, drug candidates with human safety data, and natural compounds isolated from medicinal plants. As the fish larvae are very small (3-5 mm long) and can absorb the compounds through their skin, gastrointestinal tract and gills, they are individually placed in so-called multiwell plates, in which the compound is added directly to the water. In this way many compounds can be tested simultaneously. Using the automated tracking systems, the researchers can identify those compounds that inhibit the seizure-like behavior caused by the gene mutation.

The LCSB aquatic facility currently hosts a growing number of genetically different zebrafish lines in specialized aquarium tanks. A platform manager and a team of trained researchers and technicians take daily care of the husbandry and welfare of the zebrafish. The zebrafish platform is equipped with the latest technology, such as feeding robots that assures that the adult fish are fed in optimal intervals and conditions.