Researchers have developed lab-grown “mini-brains” to better understand a brain malformation linked to drug-resistant epilepsy in children.
The MOSAIC team at the Paris Brain Institute created human cortical organoids carrying mutations in the DEPDC5 gene to model focal cortical dysplasia type II, a structural brain abnormality that can cause severe epilepsy.
The findings, published in Brain, show that both copies of the DEPDC5 gene must be inactivated to recreate the key features of the condition.
Focal cortical dysplasia type II disrupts the organisation of nerve cells in the cortex and is a known cause of drug-resistant epilepsy.
For some children affected by the condition, surgery to remove the seizure-generating area of the brain is the only treatment option, but outcomes can vary and the procedure carries risks.
The condition belongs to a group of disorders known as mTORopathies, which are driven by mutations in genes that regulate cell growth and differentiation through the mTOR signalling pathway.
These mutations can create a mosaic pattern in the developing brain, where some cells carry the mutation and others do not.
To investigate the mechanisms behind the condition, researchers collected blood cells from a young patient carrying a DEPDC5 mutation and from his unaffected brother, who acted as a control.
The cells were reprogrammed into induced pluripotent stem cells and then developed into human cortical organoids.
These tiny three-dimensional structures, only a few millimetres across, replicate key stages of human brain development.
“Mouse models of this condition existed, but they didn’t fully capture the complexity of the human patients. Human cortical development has features you simply don’t find in mice,” said lead author Marina Maletic, who worked on the project as part of her PhD.
“So, we created human mosaic organoids by mixing cells carrying two mutated copies of the DEPDC5 with cells carrying only one.
“This allows us to recreate the genetic situation observed in patients’ brains.”
The team compared healthy organoids, organoids with a single mutated copy of the gene and mosaic organoids over a six-month development period.
Only the mosaic organoids reproduced the hallmarks of focal cortical dysplasia, including abnormally large neurons, accumulated neurofilaments and overactivation of the mTOR pathway.
The findings support the “two-hit” model, first proposed in cancer genetics, which suggests two separate DNA changes are needed to trigger disease.
Professor Stéphanie Baulac, head of the MOSAIC team, said: “A single inherited mutation in the DEPDC5 gene is not sufficient to cause FCDII.
“It is the acquisition of a second spontaneous mutation — in a specific cell, at a specific moment in brain development — that sets off the pathological process.”
Maletic added: “Complete loss of DEPDC5 in a fraction of nerve cells is both necessary and sufficient to initiate the disease.
“The extent of the lesions then depends on the degree of mosaicism — in other words, on how many cells have sustained that second hit.”
Researchers also used single-cell RNA sequencing to examine the organoids at one, three and six months of development.
They found that the normal timetable of cortex development was disrupted from the first month, even in organoids with only one mutated copy of DEPDC5.
Neurons in the upper layers of the cortex appeared prematurely, with the loss of DEPDC5 abnormally activating genes involved in nerve cell maturation.
These included genes linked to the Notch and Wnt signalling pathways, which help regulate the balance between stem cell growth and differentiation into mature neurons.
The team also measured spontaneous electrical activity in six-month-old organoids using a multi-electrode array.
The mosaic organoids showed marked hyperactivity, with neurons firing more frequently and across a wider area than those in control or heterozygous organoids.
Maletic said: “You can’t really talk about epilepsy in an organoid. It’s just a model, not a real patient.
“But we do consider this abnormal electrical activity to be a correlate of epileptic seizures in humans. It rounds off our description of the pathological process.”
The researchers said the dysregulated epilepsy-associated genes identified in the study could represent new therapeutic targets.
They also said mosaic organoids could help scientists model other brain malformations that have been difficult to study because of limited access to human brain tissue.
Maletic said: “This is an excellent model that will ultimately enable precision medicine.
“By growing lab-based mini-brains from a patient’s own cells, we will be able to test multiple therapeutic options and identify which works best for that individual — without ever touching their brain.”