Mouse study shows knocking out key gene leads to autistic traits

More than 70 genes have been linked to autism spectrum disorder (ASD), a developmental disorder in which differences in the brain lead to a host of impaired behaviors, including problems with language, social communication, hyperactivity and repetitive movements. Scientists are trying to identify these specific associations gene by gene, neuron by neuron.

One such gene is astrotactin 2 (ASTN2). In 2018, researchers at The Rockefeller University’s Developmental Neurobiology Lab discovered how defects in the protein produced by the gene disrupted cerebellar circuitry in children with neurodevelopmental disorders.

Now, the same lab has discovered that completely inactivating the gene leads to several behaviors characteristic of autism. As they describe in a new paper published in PNASMice lacking ASTN2 exhibited behaviors that were markedly different from their wild-type nestmates in four main ways: they vocalized and socialized less, but were more hyperactive and repetitive in their behavior.

“All of these traits have parallels in people with ASD,” says Michalina Hanzel, first author of the study. “Alongside these behaviors, we also saw structural and physiological changes in the cerebellum.”

“This is a major discovery in neuroscience,” says Mary E. Hatten, head of the lab, whose work has focused on this brain region for decades. “It also underscores the emerging idea that the cerebellum has cognitive functions that are quite independent of its motor functions.”

An unexpected role

In 2010, Hatten’s lab discovered that proteins produced by the ASTN2 gene help guide neurons as they migrate through the developing cerebellum and shape its structure. In the 2018 study, they looked at a family in which three children had both neurodevelopmental disorders and ASTN2 mutations. They found that in a developing brain, the proteins have a similar guiding role: They keep the chemical conversation going between neurons by moving receptors off neuronal surfaces to make room for new receptors.

In the case of a mutated gene, proteins are no longer able to act and receptors accumulate, causing a traffic jam that hinders neural connections and communication. This impact has been observed in childhood conditions including intellectual disability, language delays, ADHD and autism.

The discovery is part of a growing body of evidence indicating that the cerebellum – the brain’s oldest cortical structure – is important not only for motor control, but also for language, cognition and social behavior.

For the current study, Hanzel wanted to see what effects a complete absence of the ASTN2 gene might have on cerebellar structure and behavior. Working with study co-author Zachi Horn, a former postdoctoral fellow in the Hatten lab, and with help from Shiaoching Gong of Weill Cornell Medicine, Hanzel spent two years creating a knockout mouse lacking ASTN2, then studied the brains and activity of infant and adult mice.

Behavioral parallels

The knockout mice participated in several noninvasive behavioral experiments to see how they compared to their wild-type littermates. The knockout mice showed markedly different characteristics in each of them.

In one study, researchers briefly isolated baby mice and then measured how often they called for their mothers using ultrasonic vocalizations. These sounds are a key part of a mouse’s social behavior and communication, and they’re one of the best indicators researchers have for assessing parallels with human language skills.

Wild-type pups were quick to call their mothers using complex, pitch-changing sounds, whereas knockout pups emitted fewer, shorter calls in a limited pitch range.

People with ASD often have similar communication challenges, Hanzel says. “It’s one of the most telling features, but it plays out on a spectrum,” she says. “Some people with autism don’t understand metaphors, while others repeat language they’ve heard, and still others don’t speak at all.”

In another experiment, the researchers tested how ASTN2 mice interacted with familiar and unfamiliar mice. They preferred to interact with a mouse they knew over a mouse they didn’t know. In contrast, wild-type mice always chose the social novelty of a new face.

This result is also comparable to the behavior of people with ASD, who often show reluctance toward unfamiliar environments and people, Hanzel adds. “This is a very important result because it shows that mice with the knockout mutation do not like social novelty and prefer to spend time with mice they know, which is consistent with people with ASD, who tend to enjoy novel social interactions less than familiar ones.”

In a third experiment, both types of mice were allowed to freely explore an open space for an hour. The ASTN2 mice traveled significantly further than the other mice and engaged in repetitive behaviors, such as circling, 40 percent more. Hyperactivity and repetitive behaviors are well-known features of ASD.

Poor communication between brain regions

By analyzing the brains of ASTN2 mice, they found some small but apparently important structural and physiological changes in the cerebellum. One was that large neurons called Purkinje cells had a higher density of dendritic spines, structures that are dotted with synapses that send neural signals. But they detected this change only in distinct areas of the cerebellum. “For example, we found the biggest difference in the posterior vermis region, where repetitive and inflexible behaviors are controlled,” Hanzel says.

The scientists also found a decrease in the number of immature dendritic spines called filopodia and in the volume of Bergmann glial fibers, which aid in cell migration.

“The differences are quite subtle, but they clearly affect the behavior of the mice,” Hatten says. “These changes probably alter communication between the cerebellum and the rest of the brain.”

Going forward, the researchers plan to study human cerebellar cells, which they have been developing from stem cells for half a dozen years, as well as cells with ASTN2 mutations that were donated by the family in the 2018 study.

“We would like to see if we can find parallel differences in human cells to what we found in mice,” Hatten says.

She continues: “We also want to look in detail at the biology of other genes associated with autism. There are dozens of them, but there is no common thread linking them together. We are very pleased to have been able to show in detail what ASTN2 does, but there are many more genes to study.”