The University of California, San Diego (UCSD) study uses lab-grown human brain tissue to identify neural abnormalities in Pitt-Hopkins syndrome and test gene therapy tools.
In a study published on May 02, 2022, in the journal nature communicationsScientists at the University of California San Diego School of Medicine used human brain organoids to discover how a genetic mutation associated with a severe form of autism disrupts neural development. Using gene therapy tools to restore gene function successfully rescued neuronal 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), an essential gene in brain development. Transcription factors regulate when other genes are turned on or off, so their presence or absence can have a ripple effect on the developing embryo. Still, little is known about what happens to the human brain when TCF4 it is mutated
To explore this question, the researchers focused on Pitt-Hopkins syndrome, an ASD caused specifically by mutations in TCF4. Children with the genetic condition have profound cognitive and motor disabilities and are usually nonverbal.
Pitt-Hopkins syndrome (PTHS) is a rare genetic disorder characterized by developmental delay, epilepsy, distinctive facial features, and possible intermittent hyperventilation followed by apnea. As more is discovered about Pitt-Hopkins, the developmental spectrum of the disorder is broadening to encompass difficulties with autism, anxiety, ADHD, and sensory disorders. It is related to an abnormality within chromosome 18, specifically an inappropriate expression of the TCF4 gene.
Existing mouse models of Pitt-Hopkins syndrome fail to accurately mimic the neural characteristics of patients, so the UCSD team created a human research model of the disorder. Using stem cell technology, they converted patients’ skin cells into stem cells, which then developed into three-dimensional brain organoids, or “mini-brains.”
Initial observations of brain organoids revealed a large number of structural and functional differences between TCF4-Mutated samples and their controls.
“Even without a microscope, you could tell which brain organoid had the mutation,” said study senior author Alysson R. Muotri, PhD, a professor at the UC San Diego School of Medicine, director of the Stem Cell Program at UC San Diego and a member of the Sanford Consortium for Regenerative Medicine.
The TCF4-mutated organoids were substantially smaller than normal organoids, and many of the cells were not actually neurons, but neuronal progenitors. These simple cells are meant to multiply and then mature into specialized brain cells, but in the mutated organoids, part of this process went awry.
A series of experiments revealed that the TCF4 mutation led to downstream deregulation of stockings genes and the Wnt pathway, two important molecular signals that guide embryonic cells to multiply, mature into neurons, and migrate to the correct location in the brain.
Due to this dysregulation, the neuronal progenitors did not multiply efficiently and therefore fewer cortical neurons were produced. Cells that matured into neurons were less excitable than normal and often remained clumped together rather than organized into finely tuned neural circuits.
This atypical cellular architecture disrupted the flow of neural activity in the mutated brain organoid, which the authors said would likely contribute to impaired cognitive and motor function later in life.
“We were surprised to see such important development problems at all these different scales, and it left us wondering what we could do to address them,” said first author Fabio Papes, PhD, an associate professor at the University of Campinas and a visiting scholar at UC. San Diego School of Medicine, who jointly supervised 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 retrieve the functional gene in brain tissue. Both methods effectively increased TCF4 and, in doing so, corrected the phenotypes of Pitt-Hopkins syndrome at the molecular, cellular, and electrophysiological scales.
“The fact that we can correct this gene and the whole neural system is reset, even at a functional level, is amazing,” Muotri said.
Muotri points out that these genetic interventions took place at a prenatal stage of brain development, whereas in a clinical setting, children would receive their diagnosis and treatment a few years later. Therefore, clinical trials must first confirm whether further intervention is still safe and effective. The team is currently optimizing its newly licensed gene therapy tools in preparation for such a trial, in which spinal injections of the gene vector are expected to restore TCF4 function in the brain.
“For these children and their loved ones, any improvement in cognitive-motor function and quality of life would be worth trying,” Muotri said.
“What’s really remarkable about this work is that these researchers are going beyond the lab and working hard to make these findings translatable to the clinic,” said Audrey Davidow, president of the Pitt Hopkins Research Foundation. “This is much more than a stellar academic article; it is a true measure of what well-practiced science can accomplish to improve human life.”
Reference: “Transcription factor 4 loss of function is associated with deficits in progenitor proliferation and cortical neuron content” by Fabio Papes, Antonio P. Camargo, Janaina S. de Souza, Vinicius M. A. Carvalho, Ryan A. Szeto, Erin LaMontagne, José R. Teixeira, Simoni H. Avansini, Sandra M. Sánchez-Sánchez, Thiago S. Nakahara, Carolina N. Santo, Wei Wu, Hang Yao, Barbara MP Araújo, Paulo ENF Velho, Gabriel G. Haddad and Alysson R. Muotri, May 2, 2022, nature communications.
DOI: 10.1038/s41467-022-29942-w
Coauthors 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 supported, 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 US Department of Energy Joint Genome Institute (DE-AC02-05CH11231).
Disclosures: Alysson R. Muotri is a co-founder and has an equity interest in TISMOO, a company dedicated to genetic analysis and organogenesis of the human brain.