New platform: CRISPR experimentation reveals genes, such as LNPK, that impede the development of interneurons when they are absent from forebrain assembloid models.
About 10% of genes strongly linked to autism and other neurodevelopmental conditions interfere with the development of interneurons, according to unpublished findings published on bioRxiv.
The study gleaned the effects of genes using brain ‘assembloids’ – conglomerates of dissimilar organoids that mimic developing brain tissue such as the cerebral cortex. Assembloids allow scientists to research not only the neural composition of a tissue, but also the connections and communications between different ones.
Such conglomerates are crucial for studying the development of interneurons, which originate during gestation in a region of the forebrain called the subpallium (or ventral forebrain) and then migrate to the cortex. These neurons dampen electrical activity in the brain and have been implicated in autism.
The team mutated the Assembloid genes one by one, using the gene-editing tool CRISPR. The CRISPR screening process revealed which genetic changes interfere with the development of interneurons and their journey from one tissue to another.
“We have a growing list of genes associated with [autism]and we need to find a way to map them or cluster them to see what processes they’re impacting,” says lead researcher Sergiu Pasca, professor of psychiatry and behavioral sciences at Stanford University.
SSimple cell culture systems are generally amenable to CRISPR screens, but studying the hundreds of autism-related genes in a complex cell model such as an assembloid can get “quite complicated,” says Pasca.
Pasca’s team needed to perform each of the CRISPR mutations at the same time for the results to be comparable. So they prepared around 1,000 forebrain assembloids by fusing individual cortex and subpallium organoid models, using a technique they pioneered in 2017.
After culturing the assembloids for about 10 weeks, they used CRISPR to introduce damaging mutations to knock out 425 genes expressed in the ventral forebrain that are associated with autism and other neurodevelopmental conditions. Mutations in 46 of the genes interfered with either the generation of cortical interneurons or the migration of these neurons to different parts of the forebrain.
This relatively low proportion surprised Alysson Muotri, professor of pediatrics and cellular and molecular medicine at the University of California, San Diego, who was not involved in the study. “There may be more genes that are important, but they go undetected due to the reductionist nature of screening,” he says.
The team was able to observe how one gene in particular, LNPK, modified the behavior of the endoplasmic reticulum during the migration of cortical interneurons. A 2018 report linked mutations in LNPK, which shapes the endoplasmic reticulum, to a new neurodevelopmental syndrome associated with autism. The new study helps describe the role that the endoplasmic reticulum plays in the development of interneurons.
Slow migration: In LNPK knockouts, the migration of interneurons from the forebrain to the cortex is damaged.
“Ultimately, we want to see: is each gene going to cause a different form of disease? Or are some of them going to cause similar forms of disease?” says Pasca.
JThe assembloid-CRISPR approach is “a very, very elegant design,” says Jens Schwamborn, professor of cell and developmental biology at the University of Luxembourg, who was not involved in Pasca’s research.
The downside, according to Schwamborn, is the complicated, time-consuming, and error-prone process of manually cultivating thousands of assemboids. Schwamborn’s lab creates midbrain organoids using robots, an “automated production” method that he says will be “the way forward” for cultivating assembloids.
The screening part of the experiment also has limitations and is a starting point for doing more gene-specific work, says Gaia Novarino, professor of neuroscience at the Institute of Science and Technology in Klosterneuberg, Austria, who doesn’t did not participate in the study. “Every time you do screenings, you have less understanding of what kind of mutations you’re introducing.”
LNPK is just the beginning: Pasca’s lab has begun to explore how mutations affect the other 45 genes they have linked to interneuron development. Each gene, says Pasca, is its own project.
Cite this article: https://doi.org/10.53053/MCFG3141
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