Autism in Three Dimensions: Using Brain Organoids to Study Potential Gene-Environment Interactions.

The clinical heterogeneity of autism spectrum disorder (ASD) makes it difficult to match treatments to patients.1 The unique nature of ASD in humans has also hampered the use of animal models in drug development.2,3 Brain organoids, “miniature brains” derived from human cells, have emerged as a promising alternative strategy for understanding ASD.3,4 A recently published study in Environmental Health Perspectives5 suggests these organoids may also be well suited for modeling the gene–environment interactions that have long been suspected of increasing ASD risk.6

These micrographs show 4-week brain organoids that carry the CHD8 mutation. Organoids exposed to chlorpyrifos (left) exhibited impaired length and density of neurites, shown in green, compared with untreated controls (right). Neurite outgrowth is a sensitive functional end point for demonstrating how genetics, environment, or both can impair neurodevelopment. Images: Courtesy Xiali Zhong/The Johns Hopkins University.

Lena Smirnova and colleagues compared brain organoids containing a normal version of CHD8, the gene encoding chromodomain helicase DNA binding protein 8, with those containing the high-risk ASD mutation CHD8. CHD8 is one of more than 50 genes associated with higher risk of ASD.7 The number of these genes, plus more than 50 well-supported additional genes8 with lower penetrance (i.e., a lower proportion of people with the mutation develop clinical symptoms), illustrates the genetic heterogeneity of ASD that contributes to its wide symptom variation.

At 4 weeks of differentiation, the researchers exposed both types of brain organoids to 100 µM of either the organophosphate pesticide chlorpyrifos (CPF) or its metabolite chlorpyrifos-oxon (CPO) for 24 hours. The authors noted that the high concentrations and short-term exposures “do not suggest a risk to humans in the real world.”5 Smirnova, a research associate at The Johns Hopkins University, explains, “The goal of our proof-of-principle study was to test if the pesticide perturbs similar pathways as the mutation [does], which might indicate a synergistic interaction.”

The researchers found that CPF or CPO exposure reduced CHD8 protein levels more than the mutation alone. The pesticide also exacerbated the effect of the mutation on several autism-related metabolites and neurotransmitters.

No environmental risk factor for ASD has been as strongly implicated as the gene mutations, but CPF is a known neurodevelopmental toxicant that was banned from most household uses more than 20 years ago in the United States.9 A renewed ban from use on food crops is expected to take effect in early 2022.10

“Although the CHD8 mutation is highly penetrant, and patients who carry it share certain clinical features, the severity of their symptoms can vary,” says Smirnova. “Our hypothesis is that this severity may depend on environmental toxicants like CPF, with higher exposure resulting in more severe symptoms.”

Because the current study did not use cell lines from patients with the CHD8 mutation, the researchers could not test their hypothesis directly. However, the fact that CPF or CPO exposure further reduced CHD8 protein levels in the mutant cells suggests the gene and pesticide may have similar molecular targets. In both types of brain organoids, pesticide exposure reduced neurite outgrowth, showing a disturbance of neuronal functionality. The observed reductions in CHD8 protein levels and neurite outgrowth support the possibility of more severe symptoms in CHD8 mutation carriers with higher CPF exposure, Smirnova explains.

“I find it intriguing that CPF, alone and in combination with the mutation, reduced CHD8 protein levels, and I hope the researchers will explore the biological mechanism behind this,” says Lilia Iakoucheva, an associate professor of psychiatry at the University of California, San Diego, who was not involved in the study. “Knowledge of the molecular pathways could lead to new targets for drug discovery, especially if the same pesticide affects other high-risk ASD genes in similar ways.”

The researchers also reported changes in autism-related metabolite and neurotransmitter levels due to the mutation alone, the pesticide alone, or both factors jointly. For example, only the mutant brain organoids had higher levels of the essential amino acid tryptophan after exposure to CPF or CPO. At the same time, organoids with the CHD8 mutation had an imbalance of inhibitory and excitatory neurotransmitters that was not strongly influenced by pesticide exposure. These observations, which reflect published findings in individuals with ASD,11,12 helped validate the organoid model, according to Smirnova.

Both Iakoucheva and Andrew Zimmerman—a retired professor of pediatric neurology at the University of Massachusetts who is now with Massachusetts General Hospital—view human cell–derived brain organoids as a promising model for additional studies on gene–environment interactions. “I think this kind of study is the future of ASD research, especially for drug discovery,” says Zimmerman, who was not involved in the study. He was impressed with the organoid model’s ability to reveal an imbalance of inhibitory and excitatory neurotransmitters because this imbalance has long been discussed in the field.13,14,15

“Screening patients for known ASD genes has not yet led to more effective treatments, but I can envision a future where some form of organoid testing from a patient’s own cells would achieve that goal,” says Zimmerman. “That would be an exciting move toward personalized medicine.”