How microRNAs act as a ‘blueprint’ for the developing brain

Our brains contain billions of neurons and trillions of connections, and scientists are only beginning to understand the intricate process required to build this level of complexity. This includes uncovering the role of microRNAs: small, single-stranded molecules that help regulate protein production throughout the brain and the rest of the body.

Now, Scripps Research scientists have revealed how microRNAs impact Purkinje cell development—a rare type of neuron with links to neurodevelopmental disorders. Their findings, published in Neuron, could help uncover the complicated origins of these conditions, while also shedding light on how microRNAs are involved in aging, plasticity, and other key brain processes.

“Dissecting microRNA networks in the developing brain has important implications for understanding neurodevelopmental disorders, especially in Purkinje cells, which are the most affected neuronal subtype in autism spectrum disorder,” says senior author Giordano Lippi, associate professor of neuroscience at Scripps Research.

Previous studies have suggested that microRNAs are critical for brain development, but their specific role in differentiation—the process of stem cells maturing into specialized cells—has remained unclear.

“When neurons develop, they need to at some point decide what subtype they will become, but we really didn’t know much about the blueprint that instructs this differentiation,” says Lippi. “There was a lot of evidence suggesting that microRNAs might have a very important role here, but because the tools were not good enough, we couldn’t really nail down that question until now.”

The team focused on Purkinje cells, which comprise less than 1% of cells in the cerebellum. Purkinje cells integrate information from different parts of the brain and body, enabling us to make smooth, controlled movements. They are some of the largest brain cells and have a tree-like appearance—a single axon “trunk” that supports a system of “branches” known as the dendritic arbor. Purkinje cells are also surrounded by structures called climbing fibers that wrap around the cells’ dendrites and deliver information from other parts of the brain.

To attain their large size and elaborate arbor, Purkinje cell development involves prolonged periods of growth and branching. In mice, the long process of Purkinje cell development is complete around four weeks after birth.

To investigate how microRNAs are involved in neuron differentiation, the team developed new tools that temporarily turn off microRNA function during specific developmental windows. They found that microRNAs are critical during two phases in Purkinje cell development: inhibiting microRNAs during the first week after birth resulted in Purkinje cells with less complex dendritic arbors and smaller cerebellums.

In contrast, inhibiting microRNAs during the third week after birth prevented the Purkinje cells from forming synaptic connections with climbing fibers. These findings shed light on how microRNAs control the precise timing of different aspects of Purkinje cell development that were previously thought to happen concurrently.

In collaboration with co-senior author Ian MacRae, professor of integrative structural and computational biology at Scripps Research, the team also developed a mouse model to identify which genes the microRNA molecules were targeting. Using this system, they identified two microRNAs critical for Purkinje cell development (miR-206 and miR-133) and four gene targets (Shank3, Prag1, Vash1, and En2).

When they compared the Purkinje cell microRNA-target map to a map for pyramidal neurons—a functionally different but similar-looking brain cell—they showed that the two cell types follow very different microRNA blueprints during development.

“For the first time, we were able to see that certain microRNAs are enriched in Purkinje cells but not in pyramidal neurons,” says Lippi, “and by turning these microRNAs off we show how important they are for the development of Purkinje cells’ unique morphological features.”

Notably, three of the targets involved in Purkinje cell development act as “brakes” for cell growth. When microRNA binds to these targets, it takes off the brakes, which allows Purkinje cells to grow their exaggerated dendritic arbor.

Some of these gene targets have been previously linked to neurodevelopmental disorders.

“Our results seem to suggest that there might be cases in which dysregulation of the microRNA-target networks in specific areas of the brain might be causative of some of these diseases,” says first author Norjin Zolboot, a postdoctoral fellow in the Lippi lab. “We have not actually explored any of those mechanisms, but this is something that we are looking forward to investigating in the future.”

Looking ahead, the team is also planning to use their new toolset to further investigate microRNAs’ role in development, neural plasticity, and aging.

“With these new tools, a lot of doors are opening,” says Lippi. “We have barely scratched the surface of what microRNAs are doing, but now we have a way to thoroughly investigate all of this, and I think this is a very powerful toolset that will be broadly used by the field.”

In addition to Lippi, MacRae, and Zolboot, authors of the study include Yao Xiao, Jessica Du, Marwan Ghanem, Su Yeun Choi, Miranda Junn, Federico Zampa, Zeyi Huang of Scripps Research.

Our brains contain billions of neurons and trillions of connections, and scientists are only beginning to understand the intricate process required to build this level of complexity. This includes uncovering the role of microRNAs: small, single-stranded molecules that help regulate protein production throughout the brain and the rest of the body.

Now, Scripps Research scientists have revealed how microRNAs impact Purkinje cell development—a rare type of neuron with links to neurodevelopmental disorders. Their findings, published in Neuron, could help uncover the complicated origins of these conditions, while also shedding light on how microRNAs are involved in aging, plasticity, and other key brain processes.

“Dissecting microRNA networks in the developing brain has important implications for understanding neurodevelopmental disorders, especially in Purkinje cells, which are the most affected neuronal subtype in autism spectrum disorder,” says senior author Giordano Lippi, associate professor of neuroscience at Scripps Research.

Previous studies have suggested that microRNAs are critical for brain development, but their specific role in differentiation—the process of stem cells maturing into specialized cells—has remained unclear.

“When neurons develop, they need to at some point decide what subtype they will become, but we really didn’t know much about the blueprint that instructs this differentiation,” says Lippi. “There was a lot of evidence suggesting that microRNAs might have a very important role here, but because the tools were not good enough, we couldn’t really nail down that question until now.”

The team focused on Purkinje cells, which comprise less than 1% of cells in the cerebellum. Purkinje cells integrate information from different parts of the brain and body, enabling us to make smooth, controlled movements. They are some of the largest brain cells and have a tree-like appearance—a single axon “trunk” that supports a system of “branches” known as the dendritic arbor. Purkinje cells are also surrounded by structures called climbing fibers that wrap around the cells’ dendrites and deliver information from other parts of the brain.

To attain their large size and elaborate arbor, Purkinje cell development involves prolonged periods of growth and branching. In mice, the long process of Purkinje cell development is complete around four weeks after birth.

To investigate how microRNAs are involved in neuron differentiation, the team developed new tools that temporarily turn off microRNA function during specific developmental windows. They found that microRNAs are critical during two phases in Purkinje cell development: inhibiting microRNAs during the first week after birth resulted in Purkinje cells with less complex dendritic arbors and smaller cerebellums.

In contrast, inhibiting microRNAs during the third week after birth prevented the Purkinje cells from forming synaptic connections with climbing fibers. These findings shed light on how microRNAs control the precise timing of different aspects of Purkinje cell development that were previously thought to happen concurrently.

In collaboration with co-senior author Ian MacRae, professor of integrative structural and computational biology at Scripps Research, the team also developed a mouse model to identify which genes the microRNA molecules were targeting. Using this system, they identified two microRNAs critical for Purkinje cell development (miR-206 and miR-133) and four gene targets (Shank3, Prag1, Vash1, and En2).

When they compared the Purkinje cell microRNA-target map to a map for pyramidal neurons—a functionally different but similar-looking brain cell—they showed that the two cell types follow very different microRNA blueprints during development.

“For the first time, we were able to see that certain microRNAs are enriched in Purkinje cells but not in pyramidal neurons,” says Lippi, “and by turning these microRNAs off we show how important they are for the development of Purkinje cells’ unique morphological features.”

Notably, three of the targets involved in Purkinje cell development act as “brakes” for cell growth. When microRNA binds to these targets, it takes off the brakes, which allows Purkinje cells to grow their exaggerated dendritic arbor.

Some of these gene targets have been previously linked to neurodevelopmental disorders.

“Our results seem to suggest that there might be cases in which dysregulation of the microRNA-target networks in specific areas of the brain might be causative of some of these diseases,” says first author Norjin Zolboot, a postdoctoral fellow in the Lippi lab. “We have not actually explored any of those mechanisms, but this is something that we are looking forward to investigating in the future.”

Looking ahead, the team is also planning to use their new toolset to further investigate microRNAs’ role in development, neural plasticity, and aging.

“With these new tools, a lot of doors are opening,” says Lippi. “We have barely scratched the surface of what microRNAs are doing, but now we have a way to thoroughly investigate all of this, and I think this is a very powerful toolset that will be broadly used by the field.”

In addition to Lippi, MacRae, and Zolboot, authors of the study include Yao Xiao, Jessica Du, Marwan Ghanem, Su Yeun Choi, Miranda Junn, Federico Zampa, Zeyi Huang of Scripps Research.