Group creates model that mimics malformations associated with severe epilepsy – 03/15/2022

Focal cortical dysplasia is a cerebral malformation responsible for one of the most serious types of epilepsy. The treatment of these cases remains difficult, either because of the lack of effective drugs or because of difficult access to surgery.

Now, a new human model created by researchers at Unicamp (Universidade Estadual de Campinas) using patient cells opens the possibility of testing more specific therapies and drugs.

In partnership with a group from the University of California, San Diego, scientists have created the first models of cortical organoids (3D cell culture containing specific cell types of the organ to be studied) that mimic focal cortical dysplasia, characterized by a malformation of the cortex. .

They identified mechanisms that may be involved in the emergence of the anomaly during brain formation. They were also able to obtain electrical recordings that approximate the neuronal discharge associated with epileptic seizures in humans.

You results were published in the journal brain, from Oxford Academic, one of the most relevant in clinical neurology and neuroscience. They could contribute to future work aimed at testing drugs for severe epilepsy, the kind that affects individuals who, even after two years of using adequate drugs and/or surgery, continue to have frequent seizures.

The organoids (organs developed in vitro that simulate the morphology and functioning of part of the brain) were grown from skin cells of four patients with severe epilepsy treated at Clínicas Hospital, Unicamp. These cells were reprogrammed to become pluripotent stem cells, then differentiating into neural cells.

By performing morphological, molecular and functional analyzes of the organoids, the group identified features of this cortical malformation, including changes in cell proliferation, neural network hyperexcitability, the presence of dysmorphic neurons and “balloon” cells, as well as called because of their shape. (appear hybrid, having the nucleus like that of a neuron and the cytoplasm like that of an astrocyte).

“We found a molecular alteration consistent with what is expected in cellular pathways related to neuron development and maturation. We have also demonstrated that it is possible to generate cortical organoids with electrical activity that approximates what is meant by neuronal firing associated with epilepsy. , we obtained a model similar to what we see in patients, which could, in the future, be used to screen existing drugs”, summarizes Iscia Lopes-Cendes, professor at the Faculty of Medical Sciences at Unicamp and co-author of the article.

The research was carried out during the postdoctoral work of Simoni Avansini, within the framework of the Brazilian Institute of Neuroscience and Neurotechnology (BRAINN) – a CEPIDE (Center for Research, Innovation and Diffusion) of Fapesp. The work was also funded through three other projects.

Until then, there was a limit to studies of this type of epilepsy in animal models, including rodents, because the cerebral cortex is very different from the human cortex and does not present this type of malformation.

“In the field of epilepsy, this is a very important study. Over the years there have been several attempts, with successes and errors. The result crowns Simoni’s research, which has kept something important in a researcher: perseverance,” says Lopes-Cendes, who is a senior researcher at BRAINN.

An incurable neurological disease, epilepsy affects approximately 50 million people worldwide, according to data from the WHO (World Health Organization). It is estimated that in Brazil there are 2 million records.

Patients with severe cases have between 40 and 50 seizures a day, with loss of senses and falling. Treatment is based on a combination of drugs, which does not always work.

Most drugs decrease neuron activity in a generalized way, controlling the seizures, but causing many side effects, such as drowsiness and memory impairment. Another alternative is surgery, in which the part of the brain affected by the malformation is removed.

Uncontrolled seizures, in addition to having an impact on the patient’s routine, pose a serious risk of sudden and premature death (up to three times higher than in the general population). In addition, about half of adults with epilepsy have other types of disorders, such as depression and anxiety.

“We were able to mimic the development of the neocortex and some basic features of focal cortical dysplasia. The advantage is that we obtained a human model, preserving the patient’s genetic heritage. With the organoid, it is possible to study each stage of the malformation, which begins in the development of the cortex, with repercussions on cell proliferation and differentiation,” Avansini explains to Agência Fapesp.

In the literature, it is still unclear how abnormal cortical development can contribute to the generation of epileptic seizures in dysplastic cortical tissue.

In 2018, another article published by the group, stemming from Avansini’s doctorate, suggested that a deregulation of the expression of a gene called NEUROG2, important for the process of differentiation of neurons and glial cells (astrocytes, oligodendrocytes and microglia), would have a key role in the development of the disease.

To taste

The researchers used skin cells from four patients who had not responded to drug treatment or surgery. One of them underwent three surgeries which reduced the number of seizures, but still without achieving the expected result. The other three subjects underwent two surgeries, including a child who began with seizures at 14 months of age and had part of his speech affected.

“Our data point to a molecular break at the junction of neuroepithelial cells that would affect some neurons that form the cortical plate, leading to changes in the neural network. These, in turn, would make these patients susceptible to epilepsy,” explains Professor Alysson. Muotri, of the University of California and one of the paper’s corresponding authors, in a video promoting the work.

To capture the electrical recordings, the scientists used two techniques, one of which was innovative in the field: they placed the organoid on a plate with electrodes and introduced the electrode inside the organoid. These electrodes were developed specifically for research.

The group was also able to work with three- and five-month-old organoids, which are difficult to obtain because they tend to die in a short time because they have no vascular system.

“We are driven by challenges. I had a family member with epilepsy who died from one of the seizures. When we go through this, we know exactly what is in the heart families. That’s what moves me more, the search continues,” says Avansini.

According to the researcher, the next step is to seek to better understand the formation of epilepsy and to focus on the proliferative region to understand how the cells and the circuit are formed. And, if there is a change in this step, how is it possible to interfere with the system to lead to new treatments.

Today, Avansini is a researcher at the Bioimaging Laboratory, within the National Biosciences Laboratory (LNBio), a research institute particularly focused on the use of synchrotron light. LNBio integrates the CNPEM (National Center for Research in Energy and Materials) with three other laboratories: the National Synchrotron Light (LNLS/Sirius), the National Biorenewables (LNBr) and the National Nanotechnology (LNNano).

“Our CEPID has accomplished the task of producing good science and training independent researchers who will continue to do good science”, adds Lopes-Cendes.

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