By creating individualized brain ‘organoids’ in the laboratory, researchers have revealed the role of microRNAs in brain development and demonstrated that one specific drug can reverse key cellular indicators of autism.
Scientists at Scripps Research have utilized stem cells from patients diagnosed with a rare and severe form of autism spectrum disorder, along with intellectual disability, to cultivate personalized “mini-brains” (or organoids) for further investigation. These lab-created organoids provided a deeper understanding of how a particular genetic mutation contributes to autism spectrum disorder. Additionally, they evidenced that an experimental medication named NitroSynapsin could reverse some signs of brain dysfunction typically linked with autism in these models.
“Our research demonstrates that a genetic mutation associated with autism disrupts the normal balance of brain cells during development,” explains Stuart A. Lipton, MD, PhD, Endowed Professor at the Step Family Foundation and co-director of the Neurodegeneration New Medicines Center at Scripps Research, who is also a clinical neurologist and the senior author of this new study published online in Molecular Psychiatry on September 30, 2024.
Insights from Patients
Autism spectrum disorder (ASD) is a neurological and developmental condition affecting social interaction, repetitive behaviors, and communication skills. The specific causes of ASD are not fully understood; numerous genetic variants have been linked to the disorder, but each accounts for only a small fraction of cases. For an extended period, research on ASD has relied on modeling the condition in mice or examining isolated human brain cells, neither of which accurately represents the intricate connections found in a human brain.
Lipton and his team concentrated on MEF2C haploinsufficiency syndrome (MHS), a rare and serious variant of ASD alongside intellectual disability due to a mutation in the MEF2C gene. They transformed skin cells taken from MHS patients into human stem cells using advanced stem cell techniques and cultivated them into small, millimeter-sized “mini-brain” organoids. This allowed the researchers to explore how different brain cell types interact.
“We were able to replicate vital elements of the brains of these patients to examine their electrical activity and other characteristics,” notes Lipton. “We even invited some children to our lab to see their own mini-brains, which was a very emotional experience for both the kids and their families.”
In healthy human brains and organoids, neural stem cells differentiate into nerve cells (neurons) that communicate throughout the brain, alongside various glial cells, which provide support and have recently been recognized as essential for signaling and immune functions. A healthy brain maintains a balance between excitatory neurons that enhance electrical signaling and inhibitory neurons that suppress it. Autism often causes an imbalance, leading to excessive excitation.
In organoids derived from MHS patients, the researchers discovered that neural stem cells were more likely to develop into glial cells, resulting in an abnormal surplus of glial cells compared to neurons. Particularly, the MHS organoids had a lower number of inhibitory neurons than normal. This caused heightened electrical signaling in the mini-brains, resembling conditions observed in actual human brains affected by ASD.
The Role of MicroRNA
When investigating how MEF2C mutations might contribute to this imbalance of cell types, Lipton’s group identified nearly 200 genes directly influenced by the MEF2C gene. Among these, three specific genes were particularly notable; instead of coding for DNA that leads to messenger (m)RNA and protein production, they were responsible for specific microRNA molecules.
MicroRNAs (miRNAs) are small RNA molecules that bind to DNA to regulate gene expression instead of producing proteins. Notably, this month, two scientists were awarded the 2024 Nobel Prize in Physiology or Medicine for their work on discovering miRNAs and their effects on cell development and behavior.
“In our research, certain miRNAs seem crucial in directing developing brain cells on whether to evolve into glial cells, excitatory neurons, or inhibitory neurons,” Lipton states. “Mutations in MEF2C alter the expression of these miRNAs, preventing the developing brain from forming the right types of nerve cells and establishing the correct connections or synapses between them.”
Early-stage developing brain cells from MHS patients were found to have reduced levels of three specific miRNAs. When the team added extra copies of these miRNA molecules to patient-derived brain organoids, the mini-brains developed in a more typical manner, achieving a healthier balance of neurons and glial cells.
A Possible Treatment
Since autism spectrum disorder is generally not diagnosed during the fetal stage of brain development, interventions designed to alter the initial development—such as correcting a mutated gene or introducing miRNA molecules to fix the cell type imbalance—are not feasible at this time. Nonetheless, Lipton had been working on a different drug that could help restore the balance between excitatory and inhibitory neurons, even later in development.
Recently, Lipton’s group tested this new medication, which he and his colleagues developed and patented under the name NitroSynapsin (or EM-036), to assess its ability to restore neural connections in “mini-brains” derived from cells impacted by Alzheimer’s disease.
In the study, they also evaluated whether this drug could assist in treating the MHS version of autism. Using patient-derived brain organoids, they demonstrated that NitroSynapsin could partially correct the imbalance between cell types in fully developed brain organoids. This action helped prevent excessive neuron excitability and restored the excitatory/inhibitory equilibrium in the mini-brain, thus protecting the connections between nerve cells or synapses.
Further research is necessary to determine if the drug improves symptoms for patients with MHS or impacts other forms of autism spectrum disorder not driven by MEF2C gene mutations. Lipton suggests there is a possibility of this, given that MEF2C is known to influence multiple genes associated with autism.
“We are moving ahead with testing this drug in animal models, aiming to transition to human trials soon,” Lipton remarks. “This is an exciting development.”
Co-authors of the study, titled “Dysregulation of miRNA expression and excitation in MEF2C autism patient hiPSC-neurons and cerebral organoids,” alongside Lipton include Dorit Trudler, Swagata Ghatak, Michael Bula, Maria Talantova, Melissa Luevanos, Sergio Labra, Titas Grabauskas, Emily Schahrer, Nima Dolatabadi, Clare Bakker, Parth Patel, and Rajesh Ambasudhan from Scripps; James Parker, Sarah Moore Noveral, Mayu Teranaka, Kevin Lopez, Abdullah Sultan, and Nobuki Nakanishi from the Scintillon Institute in San Diego; Agnes Chan, Yongwook Choi, Wei Lin, and Nicholas J. Schork from the Translational Genomics Research Institute in Phoenix, AZ; Riki Kawaguchi and Daniel H. Geschwind from UCLA; Pawel Stankiewicz from Baylor College of Medicine; Ivan Garcia-Bassets, Piotr Kozbial, and Michael G. Rosenfeld from UC San Diego; as well as Shing Fai Chan from Sanford Burnham Prebys Medical Discovery Institute, presently at Indiana University-Purdue University.
This research was funded by the National Institutes of Health (R35 AG071734, RF1 AG057409, R01 AG056259, R01 DA048882, R01 NS086890, and DP1 DA041722), the California Institute for Regenerative Medicine (DISC2-11070), and Autism Speaks, Inc (postdoctoral fellowship grant #11721).
