In a study published May 2, 2022 in Nature Communications, scientists from the University of California San Diego School of Medicine used human brain organoids to reveal how a genetic mutation associated with a profound form of autism disrupts neural development. Using gene therapy tools to recover gene function effectively saved neural structure and function.
Several neurological and neuropsychiatric diseases, including autism spectrum disorders (ASD) and schizophrenia have been linked to mutations in transcription factor 4 (TCF4), a gene essential in brain development. Transcription factors regulate when other genes are turned on or off, so their presence or not can have a domino effect in the developing embryo. However, little is known about what happens to the human brain when TCF4 has changed.
To explore this question, the researchers focused on Pitt-Hopkins syndrome, an ASD specifically caused by mutations in TCF4. Children with the genetic condition have profound cognitive and motor impairments and are typically non-verbal.
Existing mouse models of Pitt-Hopkins syndrome fail to accurately mimic the neural characteristics of patients, so the UC San Diego team instead created a human research model of the disorder. Using stem cell technology, they converted patients’ skin cells into stem cells, which were then developed into three-dimensional brain organoids, or “mini-brains”.
Initial observations of brain organoids revealed a number of structural and functional differences between the TCF4 – mutated samples and their controls.
“Even without a microscope, you could tell which brain organoid had the mutation,” said senior study author Alysson R. Muotri, PhD, professor at UC San Diego School of Medicine, director of UC San Diego Stem Cell program and member of the Sanford Consortium for Regenerative Medicine.
The TCF4 – the mutated organoids were substantially smaller than normal organoids, and many of the cells were not actually neurons, but neural progenitors. These simple cells are meant to multiply and then mature into specialized brain cells, but in mutated organoids, part of that process has gone wrong.
A series of experiments revealed that the TCF4 the mutation led to a downstream dysregulation of SOX genes and the Wnt pathway, two important molecular signals that drive embryonic cells to multiply, mature into neurons, and migrate to the correct location in the brain.
Due to this dysregulation, the neural progenitors did not multiply efficiently and therefore fewer cortical neurons were produced. The cells that matured into neurons were less excitable than normal and often stayed clustered together instead of organizing themselves into finely tuned neural circuits.
This atypical cellular architecture disrupted the flow of neural activity in the mutated brain organoid, which the authors say could contribute to impairing cognitive and motor function across the board.
“We were surprised to see such major development problems at all these different scales and wondered what we could do to address them,” said first author Fabio Papes, PhD, associate professor at Campinas University and visiting scholar at the UC San Diego School of Medicine, who jointly oversaw the work with Muotri. Papes has a relative with Pitt-Hopkins syndrome, which motivated him to study TCF4.
The team tested two different gene therapy strategies to recover the functional gene in brain tissue. Both methods have actually increased TCF4 levels and, in so doing, corrected the phenotypes of Pitt-Hopkins syndrome on a molecular, cellular and electrophysiological scale.
“The fact that we can correct this gene and the whole neural system is restored, even on a functional level, is amazing,” Muotri said.
Muotri notes that these genetic interventions took place in a prenatal stage of brain development, while in a clinical setting the children would receive the diagnosis and treatment a few years later. Therefore, clinical trials must first confirm whether a subsequent intervention is still safe and effective. The team is currently optimizing their recently licensed gene therapy tools in preparation for such a study, in which spinal injections of the gene vector are hoped to restore TCF4 function in the brain.
“For these children and their loved ones, any improvement in motor-cognitive function and quality of life would be worth it,” Muotri said.
“What’s really great about this work is that these researchers are going beyond the lab and working hard to make these results translatable in the clinic,” said Audrey Davidow, president of the Pitt Hopkins Research Foundation. “This is more than just a stellar academic paper; it is a true measure of what well-practiced science can accomplish to hopefully change human life for the better.”
Co-authors include: Janaina S. de Souza, Ryan A. Szeto, Erin LaMontagne, Simoni H. Avansini, Sandra M. Sanchez-Sanchez, Wei Wu, Hang Yao and Gabriel Haddad at UC San Diego; Antonio P. Camargo, Vinicius MA Carvalho, Jose R. Teixeira, Thiago S. Nakahara, Carolina N. Santo, Barbara MP Araujo and Paulo ENF Velho at the University of Campinas.
This work was funded, in part, by the National Institutes of Health (grant R01 MH123828), the Pitt Hopkins Research Foundation, the Sao Paulo Research Foundation (grants 2020 / 11451-7, 2018 / 03613-7, 2018 / 04240-0 ) and the Joint Genome Institute of the United States Department of Energy (DE-AC02-05CH11231).