Researchers at Johns Hopkins Bloomberg School of Public Health have shown in a brain organoid study that exposure to a common pesticide synergizes with a frequent autism-linked gene mutation.

The results represent one of the clearest pieces of evidence yet that genetic and environmental factors may be able to combine to disturb neurodevelopment. Researchers suspect that genetic and environmental factors might contribute to the increased prevalence of autism spectrum disorder, a developmental disorder characterized by cognitive function, social, and communication impairments.

The study’s use of brain organoids also points the way towards quicker, less expensive, and more human-relevant experimentation in this field when compared to traditional animal studies.

The brain organoid model, developed by the Bloomberg School researchers, consists of balls of cells that are differentiated from human stem cell cultures and mimic the developing human brain. The researchers found in the study that chlorpyrifos, a common pesticide alleged to contribute to developmental neurotoxicity and autism risk, dramatically reduces levels of the protein CHD8 in the organoids. CHD8 is a regulator of gene activity important in brain development. Mutations in its gene, which reduce CHD8 activity, are among the strongest of the 100-plus genetic risk factors for autism that have so far been identified.

The study, which appears online July 14 in Environmental Health Perspectives, is the first to show in a human model that an environmental risk factor can amplify the effect of genetic risk factor for autism.

“This is a step forward in showing an interplay between genetics and environment and its potential role for autism spectrum disorder,” says study lead Lena Smirnova, PhD, a research associate in the Department of Environmental Health and Engineering at the Bloomberg School.

Clinically rare as recently as 40 years ago, autism spectrum disorder now occurs in roughly two percent of live births, according to the Centers for Disease Control and Prevention.

“The increase in autism diagnoses in recent decades is hard to explain–there couldn’t have been a population-wide genetic change in such a short time, but we also haven’t been able to find an environmental exposure that sufficiently accounts for it,” says study co-author Thomas Hartung, MD, PhD, professor and Doerenkamp-Zbinden Chair in the Bloomberg School’s Department of Environmental Health and Engineering. Hartung is also director of the Center for Alternatives to Animal Testing at the Bloomberg School. “To me, the best explanation involves a combination of genetic and environment factors,” says Hartung.

How environmental factors and genetic susceptibilities interact to increase risk for autism spectrum disorder remains mostly unknown, in part because these interactions have been difficult to study. Traditional experiments with laboratory animals are expensive and, especially for disorders involving the brain and cognition, of limited relevance to humans.

Advances in stem cell methods in the past decades have allowed researchers to use human skin cells that can be transformed first into stem cells and then into almost any cell type and studied in the lab. In recent years, scientists have expanded beyond simple lab-dish cell cultures to make cultures of three-dimensional organoids that better represent the complexity of human organs.

For their study, the researchers used brain organoids to model the effects of a CHD8 gene disruption combined with exposure to chlorpyrifos. A group led by co-author Herbert Lachman, MD, professor at Albert Einstein College of Medicine, engineered the cells that make up the organoids to lack one of the two normal copies of the CHD8 gene. This modeled a substantial, but less-than-total, weakening of the CHD8 gene’s activity, similar to that seen in people who have CHD8 mutations and autism. The researchers then examined the additional effect of exposure to chlorpyrifos, which is still widely used on agricultural produce in the U.S. and abroad.

“High-dose, short-term experimental exposures do not reflect the real-life situation, but they give us a starting point to identify genetic variants that might make individuals more susceptible to toxicants,” says Smirnova. “Now we can explore how other genes and potentially toxic substances interact.”

The researchers found that the brain organoids with just one copy of the CHD8 gene had only two-thirds the normal level of CHD8 protein in their cells, but that chlorpyrifos exposure drove CHD8 levels much lower, turning a moderate scarcity into a severe one. The exposure demonstrated clearly how an environmental factor can worsen the effect of a genetic one, likely worsening disease progression and symptoms.

As part of their study, the researchers compiled a list of molecules in blood, urine, and brain tissue that prior studies have shown to be different in autism spectrum patients. They found that levels of several of these apparent autism biomarkers were also significantly altered in the organoids by CHD8 deficiency or chlorpyrifos exposure, and moreso by both.

“In this sense, we showed that changes in these organoids reflect changes seen in autism patients,” Smirnova says.

The findings, according to the researchers, pave the way for further studies of gene-environment interactions in disease using human-derived organoids.

“The use of three-dimensional, human-derived, brain-like models like the one in this study is a good way forward for studying the interplay of genetic and environmental factors in autism and other neurodevelopmental disorders,” Hartung says.

A: Illustration of the experiment. Green fluorescent proteins were introduced so that the target gene and the entire neuron could be observed. B: Each target gene was separately introduced into the cerebral cortex and dendritic spine dynamics were tracked for a period of 2 days (on the 21st and 22nd days after birth). Yellow arrows indicate newly formed spines and red arrowheads indicate spine elimination. C: The quantified results of B in graph form. D: Spine morphology classification data on the spines that formed when Ndn was introduced. Filopedia, a type of immature spine formation, significantly increased upon Ndn introduction. CREDIT Takumi et al., Nature Communications, 2021

A research group including Kobe University’s Professor TAKUMI Toru (also a Senior Visiting Scientist at RIKEN Center for Biosystems Dynamics Research) and Assistant Professor TAMADA Kota, both of the Physiology Division in the Graduate School of Medicine, has revealed a causal gene (Necdin, NDN) in autism model mice that have the chromosomal abnormality (*1) called copy number variation (*2).

The researchers hope to illuminate the NDN gene’s molecular mechanism in order to contribute towards the creation of new treatment strategies for developmental disorders including autism.

These research results were published in Nature Communications on July 1, 2021.

Main Points

• The research group identified Ndn as a causal gene of autism by conducting a screening based on synaptic expression in an animal model of the disorder (15q dup mouse).
• The Ndn gene regulates synapse development during the developmental stage.
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Research Background

Even though the number of patients diagnosed with autism (autism spectrum disorder) has been greatly increasing, many aspects of this developmental disorder are still not well understood. Its causes are divided into genetic factors and environmental factors. Within these genetic factors, particular copy number variations have been found in autistic patients; for example, chromosome 15q11-q13 duplication. These abnormalities in the 15q11-q13 region are divided into maternally derived and paternally derived chromosomal duplication cases. It is understood that the Ube3a gene drives maternally derived chromosomal duplication. However, it is not known which gene is vital for paternally derived duplication.

This research group previously succeeded in developing a mouse model of 15q11-q13 duplication (15q dup mouse). Using this mouse model, they identified numerous abnormalities in paternally derived chromosomal duplication cases, including autism-like behaviors, and abnormalities in dendritic spine (*4) formation. However, the researchers were unable to identify which gene is responsible for autism-like behavior because this region contains many non-coding RNA molecules and genes that code proteins.

Research Methodology

In 15q dup mice, there are a great number of genes as the duplication extends to the 6Mb region. Previous research showed that behavioral abnormalities were not induced by maternally derived chromosomal duplication, therefore around 2Mb was excluded. As for the remaining 4Mb, the researchers first created a new 1.5Mb duplication mouse model and investigated behavior abnormalities. From the results, they were unable to identify any autism-like behavioral abnormalities in 1.5Mb duplication mice. Consequently, the researchers excluded this 1.5Mb, leaving them with three protein-encoding genes as possible candidates.

Next, these three genes were individually introduced into the cerebral cortex of mice via in utero electroporation (*5). The researchers measured the spine turnover rate (formation and elimination of dendritic spines over a 2 day period) in vivo using a two-photon microscope (*6) and discovered that the number of spines drastically increased when the Ndn gene was introduced (Figure 1A-C). Furthermore, morphology classification of these spines indicated that the majority were immature. This reveals that the Ndn gene regulates the formation and maturation of dendritic spines during the developmental stage (Figure 1D).

Using CRISPR-Cas9 (*7), the researchers subsequently removed the one copy of the Ndn gene from the 15q dup mouse model to generate mice with a normalized genomic copy number for this gene (15q dupΔNdn mouse). Using this model, they demonstrated that the abnormalities observed in 15q dup mice (abnormal spine turnover rate and decreased inhibitory synaptic input) could be ameliorated (Figure 2).

Lastly, the researchers investigated whether the previously observed autism-like behaviors in 15q dup mice (including increased anxiety in a new environment, reduced sociability and increased perseveration) were evident in 15q dupΔNdn mice. They showed that in the majority of behavioral test results for 15q dupΔNdn mice, abnormal behaviors related to sociability and perseveration were ameliorated (Figure 3).

Further Research

This research study revealed that in 15q dup autism model mice, the NDN gene does not only play an important role in autism-like behaviors, but also affects aspects such as excitation/inhibition imbalance in synaptic dynamics and the cerebral cortex. Next, the research team hopes to clarify the NDN gene’s functions. By artificially regulating these functions or identifying and controlling their downstream factors, the researchers hope to understand the onset mechanism of developmental disorders like autism, and develop new treatment strategies.

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Glossary

1. Chromosomal abnormality: Chromosomes are structures that contain genes and are located inside the cell nucleus. Abnormalities such as duplications or deletions in specific regions of the chromosome are often seen in those with autism.

2. Copy number variation: A chromosomal abnormality where the number of copies of a region on the genome is abnormal (duplications or deletions). Normally there should be 2 copies (diploid). If there are 3 copies or just one copy, then this is a copy number variation.

3. Synapse: A junction between two nerve cells (neurons), which is involved in neuronal activities such as transmitting signals.

4. Dendritic spine: These are protrusions on the dendrites of excitable neurons that receive input from synapses.

5. in utero electroporation: A method of introducing outside DNA into the brain. DNA is injected through the wall of the uterus and is induced to enter the cells using electric waves.

6. Two-photon microscope: A microscope with powerful laser, which enables the brains of mice to be observed while they are still alive. In this study, it was used to track changes in dendritic spines over a 2 day-period.

7. CRISPR-Cas9: Technology that enables a specific region of a genome to be precisely cut and altered. This genome editing method won the 2020 Nobel Prize in Chemistry. In this study, it was used to remove the Ndn gene from the genome.

Coronal section of a mouse brain, with several major axonal tracts stained in green. Image courtesy of Dr. Ahlem Assali. CREDIT Dr. Ahlem Assali, Medical University of South Carolina

After reviewing a database of gene mutations in children with autism , a team of Medical University of South Carolina (MUSC) researchers decided to study a specific gene mutation that likely caused autism in a girl. They demonstrated that the mutation was damaging to the gene, and that female, but not male, mice lacking a working copy of the gene also showed autism -associated symptoms. Better understanding the interplay between genetics and sex in autism could set the stage for developing sex-specific treatments for autism.

The MUSC team was led by Christopher Cowan, Ph.D., the William E. Murray SmartState Endowed Chair in Neuroscience and chair of the Department of Neuroscience, and Ahlem Assali, Ph.D., research assistant professor in the same department. Their findings are published in Nature Neuropsychopharmacology.

One in 54 children is diagnosed with an autism . Of the children with autism, four boys are diagnosed for every girl. Individuals with autism typically have deficits in communication and social interaction and exhibit restricted, repetitive patterns of behavior, activities, or interests. Many people with autism also present with associated symptoms, such as hyperactivity, attention deficits, epilepsy and intellectual abilities that can range from severely disabled to gifted.

Cowan and Assali investigated the effect of a mutation in the gene, EPHB2, detected in a female patient with autism. EPHB2 is important for forming connections, or synapses, in the brain. The patient had a version of EPHB2 that caused the protein to be cut short. “It’s as if a sentence had a period in the middle instead of the end,” said Cowan. The shortened protein can no longer serve its function, leaving this autism individual with less functional protein than neurotypical people.

To confirm that this gene can cause autism, Cowan and Assali created mice that had only one of two working copies of EPHB2. They found that these animals showed repetitive behaviors, hyperactivity and learning and memory problems as well as changes in brain cell function.

Cowan and Assali went a step further and divided the animals based on sex. They did this because the child with ASD and the EPHB2 mutation was a female. They found that the female mice showed much stronger behavior symptoms and brain cell dysfunction than the male mice. Understanding the interplay between genetics and biological sex could be important for understanding autism risk and eventually for developing therapeutics.

“We know that 80% to 90% of autism risk is genetic, but this is a very clear-cut case where the gene and the sex of the animal are interacting to alter neurotypical development,” said Cowan.

Historically, autism has been diagnosed mostly in boys, so research on autism has often been biased toward male subjects. The work of Cowan and Assali highlights the importance of sex-specific differences in autism and the need to examine those differences in research studies that include both sexes. This could set the stage for developing sex-specific treatments for autism .

“That’s the only way we’re going to start to change research inequalities that have happened in the past,” said Assali.

In future studies, Cowan and Assali hope to explore more deeply the mechanisms of EPHB2 actions in the developing brain. They want to understand why this gene causes symptoms predominantly in female subjects and how hormones might affect autism risk. Their aim is to improve the understanding of the interplay between genetics and biological sex in autism, with a view to informing future personalized, sex-specific treatments for autism.