Although many forms of autism spectrum disorders (ASD) are thought to have genetic causes, it remains unclear how the identified genes function at cellular and molecular level. Scientists at the Institute of Science and Technology (IST) Austria have studied a high-risk gene and discovered its important role during a critical phase of brain development.
Within the European Union alone, about three million people are affected by autism spectrum disorder (ASD). Some are mildly affected, allowing them to lead independent lives, while others have severe disabilities. What the different forms have in common is difficulty interacting and communicating socially and repetitive, stereotypical behaviours. Mutations in hundreds of different genes are associated with ASD. One of these is called cullin 3 - a high-risk gene. A mutation in this gene almost certainly leads to some form of ASD. But how exactly does this gene affect the brain? To find out more, Jasmin Morandell and Lena Schwarz, PhD students in Professor Gaia Novarino's research group, worked with mice whose Cullin 3 gene had been partially switched off and compared them with their healthy siblings.
In a series of behavioural and movement tests, the team wanted to find out whether the altered mice could reproduce characteristics of patients with this form of autism and thus be used as model organisms. In one of these tests, called the "Three Chamber Sociability Test", a mouse was able to explore three adjacent chambers of a box connected by small doors. The scientists then placed two other mice in the outer boxes: one was already familiar to the mouse studied, the other had never met it before. "Healthy mice usually prefer the new mouse to the mouse they already know," explains Jasmin Morandell, co-first author of the study. However, the mouse with the altered cullin 3 gene showed no signs of this. In addition, the mice had motor coordination deficits as well as other cognitive impairments typical of ASD. With the help of this mouse model, the team was finally able to find out how the observed changes develop.
When the researchers examined the mouse brain, they noticed a slight but constant change in the location of some brain cells. These so-called neurons or nerve cells develop in a special region in the brain. From there, they migrate towards the top layers until they reach their designated place in the cerebral cortex. This process is very sensitive - if the cells migrate a little too slowly or a little too fast, it changes the composition of the cerebral cortex. By labelling the migrating neurons, the scientists were able to track their movements. "We were able to observe migration deficits - the neurons remain in the lower cortex layers," describes Lena Schwarz, the second co-first author of the study. But why don't the cells move as they should?
The answer lies in the important role that Cullin 3 plays at the end of life of proteins. When their time comes, the Cullin 3 gene marks them for degradation - a process that must be tightly regulated to prevent proteins from accumulating. To find out which proteins are misregulated when the Cullin 3 gene is defective, Morandell and Schwarz systematically analysed the protein composition of the mouse brain. "We looked at proteins that accumulate in the mutant brain and came up with a protein called plastin 3. Then Gaia came across a poster about the work of the Schur group at IST Austria - that's when things got really exciting," says Jasmin Morandell. "They had been independently studying how plastin 3 regulates cell motility and had come up with results that complemented ours, so we started working together," recalls Professor Gaia Novarino.
It turned out that the protein plastin 3, previously unknown in the context of neuronal cell migration, actually plays a crucial role in this process. "When the Cullin 3 gene is deactivated, the plastin 3 protein accumulates, causing cells to migrate more slowly and over shorter distances. This is exactly what we observed in the cerebral cortex of the modified Cullin 3 mice," PhD student Lena Schwarz tells us.
All this takes place at a very early stage of brain development, around mid-pregnancy - long before anyone would notice any difference in the foetus. "Determining this critical window of time during brain development could be extremely important in refining treatment for patients with certain forms of ASD," explains Novarino, who is working to better diagnose ASD and thus better treat people. "Further research into plastin 3 could pave the way for new therapeutics. Inhibiting the accumulation of this protein could possibly alleviate some of the symptoms that patients have," says Schwarz.
"We now know that a defective Cullin 3 gene leads to increased plastin 3 levels. This close connection shows that the plastin 3 level could be an important factor in controlling the movement of the cells," says Jasmin Morandell, who recently graduated and will use her expertise to research Huntington's disease in the future. Lena Schwarz will now turn her attention to other ASD high-risk genes at IST Austria to see how the degradation of other proteins might be linked to ASD. For this study, the Novarino group teamed up with the Danzl and Schur groups and a colleague from the University of Rome. "Completing this extensive study in about two and a half years despite the pandemic was only possible with the support of our neighbours at IST Austria," says Novarino, praising the multidisciplinarity at the Institute.
Jasmin Morandell, Lena A. Schwarz et al. 2021. Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development. Nature Communications.