Hyperactivity, repetitive actions, and difficulties with language were all observed in the mice, revealing an unexpected function of the cerebellum in autism.
Over 70 genes have been associated with autism spectrum disorder (ASD), a developmental disorder where brain differences result in a variety of altered behaviors, including language challenges, social communication difficulties, hyperactivity, and repetitive habits. Researchers are working to uncover these specific connections one gene at a time, and neuron by neuron.
One such gene is Astrotactin 2 (ASTN2). In a study from 2018, scientists from the Laboratory of Developmental Neurobiology at Rockefeller University learned how abnormalities in the protein produced by this gene caused disruptions in the cerebellar circuitry of children with neurodevelopmental disorders.
Now, the same laboratory has discovered that completely removing this gene results in multiple key behaviors typical of autism. According to their recent publication in PNAS, mice that were missing ASTN2 exhibited noticeably different behaviors than their normal counterparts in four significant ways: they communicated and socialized less, but showed increased hyperactivity and repetitive behaviors.
“All of these behaviors find parallels in individuals with ASD,” remarks Michalina Hanzel, the lead author of the study. “In addition to these behaviors, we observed both structural and physiological changes in the cerebellum.”
“This is a major discovery in neuroscience,” states lab director Mary E. Hatten, who has dedicated decades to studying this brain region. “It also emphasizes the emerging concept that the cerebellum serves cognitive roles that are largely distinct from its motor functions.”
An Unexpected Role
In 2010, Hatten’s team discovered that proteins derived from the ASTN2 gene assist in guiding neuron migration during the cerebellum’s development and structural formation. In the 2018 investigation, the researchers studied a family with three children affected by neurodevelopmental disorders and ASTN2 mutations. They determined that, in a developed brain, these proteins play a similar guiding function: they facilitate ongoing chemical communication between neurons by relocating receptors, making space for new ones. When the gene is mutated, the proteins fail to function, causing receptor accumulation which creates a blockage that impedes neuronal connections and communication. This dysfunction was evident in the children’s conditions, which included intellectual disabilities, language delays, ADHD, and autism.
This finding was part of a growing body of evidence showing that the cerebellum—one of the brain’s oldest cortical structures—is crucial not only for motor action but also for functions related to language, cognition, and social behavior.
In this latest study, Hanzel aimed to understand how the complete absence of the ASTN2 gene might affect both the structure of the cerebellum and the resultant behaviors. Working alongside co-author Zachi Horn, a former postdoctoral fellow at Hatten’s lab, and with assistance from Shiaoching Gong of Weill Cornell Medicine, Hanzel spent two years developing a knockout mouse that lacked ASTN2. They then analyzed the brains and behavior of both juvenile and adult mice.
Behavioral Parallels
The knockout mice underwent various noninvasive behavioral tests to compare their characteristics with their wild-type counterparts. The knockout mice displayed distinctly different behaviors across all experiments.
In one test, researchers briefly separated the baby mice and measured how often they called out for their mothers using ultrasonic vocalizations. These calls are crucial to a mouse’s social behavior and communication and serve as effective indicators of language abilities in humans.
The wild-type pups quickly called out to their mothers using complex sounds that varied in pitch, while the knockout pups produced fewer and shorter calls within a limited pitch range.
Similar communication difficulties are prevalent among individuals with ASD, Hanzel notes. “It’s a very telling characteristic, though it exists along a spectrum,” she explains. “Some autistic individuals may struggle with metaphor, others may echo phrases they’ve heard, and some may not speak at all.”
In another experiment, researchers observed how ASTN2 mice interacted with known and unknown mice. The knockout mice preferred to engage with familiar mice rather than new ones, while wild-type mice favored interacting with unfamiliar peers.
This behavior also mirrors that of humans with ASD, who often show reluctance towards new environments and people, Hanzel adds. “This is a significant finding, as it illustrates that knockout mice are less inclined toward social novelty and prefer spending time with familiar subjects, much like individuals with ASD who prefer familiar social interactions.”
In a further experiment, both mouse types were given the freedom to explore an open area for an hour. The ASTN2 mice moved significantly further than their counterparts and exhibited repetitive actions, such as circling in place, 40% more frequently. Both hyperactivity and repetitive actions are well-established signs of ASD.
Miscommunication Between Brain Regions
Upon examining the brains of the ASTN2 mice, researchers noted a few small but impactful structural and physiological modifications in the cerebellum. One notable alteration was that Purkinje cells—large neurons—exhibited a higher density of dendritic spines, which are loaded with synapses that transmit neural signals. However, this particular change was only observed in specific regions of the cerebellum. “For instance, we identified the most significant difference in the posterior vermis area, which regulates repetitive and rigid behaviors,” says Hanzel.
The team also discovered a reduction in both the number of immature dendritic spines known as filopodia and the volume of Bergmann glial fibers that assist with cell migration.
“The changes are subtle but clearly influence mouse behavior,” Hatten explains. “These alterations likely affect communication between the cerebellum and other brain regions.”
In the future, the researchers aim to investigate human cerebellar cells, which they have been developing from stem cells for the past six years, as well as cells with ASTN2 mutations contributed by the family involved in the 2018 study.
“We hope to identify similarities between our mouse findings and human cells,” Hatten states.
She adds, “We also want to explore the intricate biology of other autism-associated genes. While there are many, a unifying theme among them has not been established. We are thrilled to have detailed what ASTN2 does, but there remains a multitude of genes to explore.”
