A team led by Brazilian scientists at Unicamp (University of Campinas State) and the University of San Diego in California, USA, presented the mechanism that causes Pitt-Hopkins Syndrome, a neuropsychiatric disorder characterized by Autism Spectrum Disorder (ASD). . In addition, researchers were able to reverse the evolution of the syndrome in laboratory models, opening up new treatment options.
The FAPESP-sponsored work was published in the magazine this Monday (02/05) Nature Communications.
“For most cases of ASD, it is not known which gene causes the condition to be mutated. So it also happens in most neuropsychiatric disorders, such as schizophrenia, depression, and bipolar disorder. TCF4. But until then, its molecular mechanisms were not known, which is different in the cells of the nervous system of patients with mutations, ”says Fabio Papes, a professor at the Institute of Biology (IB-Unicamp) and one. study coordinators.
Papes and the team led by Professor Alysson Muotri of the University of California, San Diego, however, went beyond finding the mechanism that causes the situation.
Scientists tested ways to interfere with the evolution of the situation and managed to reverse the effects of the mutation. The success of the experiments has led to the development of both drugs and gene therapy.
Pitt-Hopkins syndrome has cognitive deficits, profound motor retardation, impaired functional speech, and respiratory abnormalities, among others. It was described in 1978, but its causal gene was not known until 2007. The mutation in the gene is estimated TCF4 It occurs in one in 35,000 births.
small brains
Because the syndrome does not develop in humans as well as in humans, it is not feasible to study it in animals. So the researchers used so-called brain organoids, a set of human cells that look like a miniature brain growing and developing in the lab, but with no vascularization and fewer cell types.
“The organoid of the brain is a more representative model than any other for the study of dysfunctions of the central nervous system. In this case, the resulting cell is from the sick cell itself. In addition, the organoid is three-dimensional, so its functioning is closer. , Papes explained.
Organoids were derived from skin biopsies of patients with the syndrome, obtained from patients recruited at Unicamp and the United States, who served as controls in addition to their parents.
Cells were grown to produce so-called fibroblasts, which transform into pluripotent stem cells and, in turn, can produce many types of human cells. In this case, they created neurons, progenitor cells in the central nervous system, and organoids in the brain.
While the cells of the patient’s parents formed normally developing organoids, the number of patients with the syndrome grew less due to the reduced replication of the cells caused by the mutations and the deterioration of the neurogenesis itself. That is, the formation of neurons was impaired as a result of the mutation.
In addition, organoid neurons are mutated TCF4 they were smaller and had less electrical activity compared to control organoids. It is well known that communication between these cells is carried out by electrical impulses, without which they cannot fulfill their functions. This finding may therefore explain many of the clinical features of patients.
The results were similar to those obtained in the brain tissue of a patient who died for other reasons, which reinforces the effects obtained with organoids. It was the first study to investigate the brain of a person with Pitt-Hopkins syndrome.
“Head access post mortem it was essential to validate some of the results obtained with brain organoids. Seeing similar features between the organoid produced in the laboratory and the brain shows how important this technology is, ”says Muotri.
gene therapy
Changes caused by mutations in a gene TCF4 the researchers looked for ways to correct it and thus implement what they call the concept of proof of what would be a treatment.
Three interventions were tested, one using a gene editing technique known as CRISPR-Cas9, the founders of which won the Nobel Prize in Chemistry in 2020.
For the strategy involving CRISPR, a final version of the technique was used to begin to express much more protein in the functional copy of the gene in the dysfunction cell, compensating for the copy caused by the mutation that causes Pitt-Hopkins syndrome.
In another intervention, using a different technique, the scientists inserted an extra copy of the gene, and began to perform normal gene functions, compensating for the mutated copy.
“Our genome has two copies of each gene. It’s the one that causes Pitt-Hopkins syndrome TCF4 No work. Inserting a third copy or a single working copy may indicate more protein to compensate for the error, ”says the researcher.
The organoids that underwent the interventions began to grow normally and increased the proliferation of the progenitor cells, which produce different types of cells in the brain, including neurons.
“While this disorder is considered rare, there are others that involve mutations in the same gene. That’s why what we’ve found here may be applied in the future to disorders like schizophrenia, for example,” says Papes.
The third intervention was the application of a drug used in tumor cell research. Known by the acronym CHIR99021, it activates the cellular signaling pathway known as Wnt, which has been extensively researched in the context of cancer and has been found by authors to be altered by gene mutations. TCF4.
In drug-treated dysfunctional cells and organoids, there was an increase in the improvement and size of certain molecular indicators (in the case of organoids). The results pave the way for the development of similar drugs that can treat dysfunction, as CHIR99021 cannot yet be used in humans.
“This drug – treated pathway is just one of those mutations TCF4. The advantage of a gene therapy over a pharmacological treatment is that it solves the problem at the outset. However, the search for new medicines is also promising, ”says Papes.
The research should now advance to preclinical and clinical research. The researchers closed a partnership with a company that specializes in gene therapy because it is licensing the technology used in the experiments so that it can be tested in humans in the future.