Study opens up new treatment possibilities for autism

A group led by Brazilian scientists from the State University of Campinas (Unicamp) and the University of California San Diego, in the United States, unveiled the mechanism that causes Pitt-Hopkins syndrome, a neuropsychiatric disorder that has characteristics of autism spectrum disorder (ASD). . In addition, the researchers managed to reverse the evolution of the syndrome in laboratory models, opening up new possibilities for treatment.

The work, supported by Fapesp, was published this Monday (05/02) in the journal Nature Communications.

“For most cases of ASD, it is not known which gene causes the condition when mutated. So it is also for most neuropsychiatric disorders such as schizophrenia, depression and bipolar disorder. Pitt-Hopkins syndrome, in turn, has a mutation in the TCF4 gene as its origin. But, until then, its molecular mechanisms were not known, that is, what is different in the cells of the nervous system of patients with the mutation”, says Fabio Papes, professor at the Institute of Biology ( IB-Unicamp) and one of the study coordinators.

The group led by Papes and University of California San Diego professor Alysson Muotri, however, went beyond discovering the mechanism causing the condition.

Scientists tested ways to interfere with the evolution of the condition and managed to reverse the effects caused by the mutation. The success obtained in the experiments paves the way for the development of both drugs and gene therapy.

Pitt-Hopkins syndrome is characterized by cognitive deficit, profound motor delay, lack of functional speech and respiratory abnormalities, among others. It was described in 1978, but its causative gene was only known in 2007. It is estimated that the mutation in the TCF4 gene occurs in one in every 35,000 births.

mini brains

Since the syndrome does not develop in mice in the same way as in humans, study in animals is not feasible. So the researchers used so-called brain organoids, a cluster of human cells that grow in the laboratory and resemble a miniature developing brain, but without vascularization and with fewer cell types.

“The brain organoid is a more representative model than any other to study central nervous system dysfunctions. In this case, the cell obtained is from the patient himself. In addition, the organoid is three-dimensional, therefore, its functioning is closer to the reality of the than cells grown in plates, which grow in only two dimensions”, explains Papes.

The organoids were generated from skin biopsies of patients with the syndrome, obtained from patients recruited at Unicamp and in the United States, in addition to their parents, who served as controls.

The cells were grown to extract so-called fibroblasts, which are transformed into pluripotent stem cells, which in turn can generate many types of human cells. In this case, they gave rise to neurons, central nervous system progenitor cells, and brain organoids.

While the cells of the patients’ parents formed organoids that developed normally, those of the patients with the syndrome grew less, as a result of the reduced cell replication caused by the mutation and an impairment of neurogenesis itself. That is, the generation of neurons was impaired because of the mutation.

In addition, neurons from organoids with the TCF4 mutation were fewer and had lower electrical activity compared to control organoids. It is known that the communication between these cells is made from electrical impulses, without which they cannot perform their functions. This finding, therefore, may explain many clinical characteristics of the patients.

The results were similar to those obtained in brain tissue from a patient with the condition, who died for other reasons, which reinforces the conclusions obtained with the organoids. The study was the first known to study the brain of a person with Pitt-Hopkins syndrome.

“Access to the post-mortem brain was essential for us to validate some of the results obtained with brain organoids. The fact that we saw similar characteristics between the organoid created in the laboratory and the brain shows how relevant this technology is”, says Muotri.

gene therapy

Once the changes caused by the mutation in the TCF4 gene were observed, the researchers looked for ways to correct it and thus carry out what they call a proof of concept of what a treatment would be.

Three interventions were tested, one using the gene editing technique known as CRISPR-Cas9 – whose creators won the Nobel Prize in Chemistry in 2020.

For the strategy involving CRISPR, a recent version of the technique was used to make the functional copy of the gene existing in the dysfunctional cell start to express much more protein, compensating the copy affected by the mutation that causes Pitt-Hopkins syndrome.

In another intervention, using a different technique, the scientists inserted an extra copy of the gene, which began to carry out normal gene functions, compensating for the mutated copy.

“Our genome has two copies of each gene. What causes Pitt-Hopkins syndrome is that one of the copies of TCF4 doesn’t work. Inserting a third copy or making the only working copy express more protein to compensate for the defective one. can solve the problem”, says the researcher.

The organoids that underwent the interventions began to grow normally and had an increase in the proliferation of progenitor cells, which in the brain give rise to different types of cells, including neurons.

“Although this disorder is considered rare, there are others that involve mutations in the same gene. Therefore, what we discovered here may, in the future, be applied to disorders such as schizophrenia, for example”, says Papes.

A third intervention was the application of a drug used in studies with tumor cells. Known by the acronym CHIR99021, it activates a cellular signaling pathway known as Wnt, which has been extensively studied in the context of cancer and which the authors discovered is also altered by mutations in the TCF4 gene.

In dysfunctional cells and organoids treated with the drug, there was an improvement in some molecular indicators and an increase in size (in the case of organoids). The results pave the way for the development of similar drugs that can treat the dysfunction, as CHIR99021 cannot yet be used in humans.

“This drug-treated pathway is just one of those altered by the mutation in the TCF4 gene. The advantage of a gene therapy over a pharmacological treatment is that it would solve the problem at its source. However, the search for new drugs is also promising”, says Papes.

The research must now advance to preclinical and clinical studies. The researchers closed a partnership with a company specializing in gene therapy, which is licensing the technology used in the experiments so that it can be tested in humans in the future.

The work was also supported by Fapesp through grants for a master’s degree to José Ricardo Teixeira Júnior and a doctorate to Antônio Camargo, both graduate students at IB-Unicamp.

The research was also supported by the National Council for Scientific and Technological Development (CNPq), the National Institutes of Health (NIH), in the United States, and the Pitt-Hopkins Research Foundation.