Researchers have built a library of mouse embryonic stem cell models to investigate how certain genetic changes linked to autism spectrum disorders affect brain development. The work has been made available to other researchers for free.
Copy-number variations (CNVs) – sections of DNA that are deleted or duplicated – have been strongly associated with ASD and other neuropsychiatric conditions. However, they often encompass multiple genes, making it difficult to pinpoint how they contribute to disease. To tackle this, the research team used CRISPR-Cas9 genome editing with a high-efficiency “next-generation chromosome engineering” method to create 63 embryonic stem cell (ESCs) lines, each carrying a CNV corresponding to one found in people with ASD.
Twelve representative CNVs were then selected for detailed functional analysis. The team differentiated the cells into neurons and other brain cell types, and then examined them using single-cell RNA sequencing and physiological assays. The work identified cell-type-specific vulnerabilities, with notable changes in glutamatergic and GABAergic neurons — key players in excitatory and inhibitory brain signaling.
One common feature across the neuronal types was reduced expression of Upf3b, a gene involved in translation termination and nonsense-mediated mRNA decay, a cellular process that removes faulty RNA. Changes in genes controlling protein synthesis, including elements of the mTOR signaling pathway, were also observed. The authors noted that disruptions to these pathways have been linked to ASD in previous studies, suggesting a converging mechanism.
They were also able to develop a mouse model based on one CNV: the 15q13.3 deletion. In humans, this is associated with social deficits and other traits seen in ASD. These mice displayed similar behavioral features, providing in vivo support for the relevance of the ESC-derived cell lines.
The ESC library is available through the RIKEN BioResource Center and could be beneficial for both in vitro studies and for generating animal models.
To find out more about the research, we also reached out to Toru Takami from Kobe University.
How did you become interested in autism research?
When I became a principal investigator, I wanted to embark on something new. Having a background in molecular biology and a deep interest in the brain and mind, I initially considered creating a mouse model based on biological abnormalities observed in patients with psychiatric disorders. We first attempted to develop a schizophrenia model, but the project did not progress as hoped…
Eventually, we shifted our focus to autism. At that time – around the end of the 20th century – biological research on autism was just beginning in the US, and no one in Japan was working in this area.
Over the course of your career, how has the field of autism research changed – scientifically and socially?
When I began autism research in the early 21st century, there was still considerable doubt as to whether autism could be understood or analyzed biologically. In Japan, at least, autism research was considered part of psychology rather than a biological field. Most people did not believe that autism was a genetic disorder.
Even today, there are still some who question whether autism should be classified as a biological disorder, in contrast to many other common medical conditions.
What inspired this project involving stem cell lines? And how did you come to use CRISPR?
Our first autism model was based on CNV and was generated using chromosome engineering with the Cre-loxP system and embryonic stem cell technology. After we published this work in Cell in 2009, numerous clinical cases – nearly 100 – reporting CNVs in individuals with autism were subsequently identified.
While we went on to develop a second and third mouse model, the process was time-consuming. Eventually, I decided to return to cellular models. Although many researchers in Japan were using iPS cell technology, I didn’t have direct access to patient samples, so we opted to work with embryonic stem cell lines. We initially used TALENs, but after reading Feng Zhang’s 2013 Cell paper, we decided to switch to CRISPR.
The work has taken over a decade. What were the biggest challenges?
We aimed to collect all known cases of CNVs. A single laboratory like ours cannot carry out projects like this – they require a consortium involving multiple contributors. And postdocs and scientists are generally less interested in simply gathering biological resources! Technicians and undergraduate students undertook much of the work involved in generating cell models.
You observed that neurons with certain autism-linked mutations failed to eliminate misshapen proteins; can you elaborate on the mechanisms involved and why that might be significant?
In addition to transcription, translation is also believed to play a role in the pathology of autism. Unfortunately, the underlying mechanisms remain unclear. This will be a focus in our future research.
How could these cell lines help accelerate drug discovery for autism?
The library we have developed includes mouse embryonic cell lines, which can enable the generation of mouse models. Once we transition to human ES cell lines, we can use differentiated cells or organoids – similar to iPS cells – for drug discovery applications.
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