LA JOLLA, CA The human brain is an incredibly intricate organ, composed of billions of neurons interconnected through trillions of synaptic connections. The formation of such an elaborate network during development is a complex and highly regulated process that has long intrigued neuroscientists. A key part of this process is understanding how different molecular components contribute to neural differentiation, growth, and connectivity. Among these molecular regulators are microRNAs small, single-stranded RNA molecules that do not code for proteins but instead play a crucial role in regulating protein synthesis in cells throughout the body, including the brain.

In an exciting breakthrough, scientists at Scripps Research have uncovered a fundamental role for microRNAs in the development of a specific type of neuron known as Purkinje cells. These large, uniquely shaped neurons reside in the cerebellum and play an essential role in coordinating movement, integrating sensory information, and even contributing to cognitive functions. More significantly, Purkinje cells have been implicated in neurodevelopmental disorders such as autism spectrum disorder (ASD), making them a prime target for research aimed at uncovering the underlying causes of these conditions.

The findings, published in *Neuron* on April 2, 2025, provide a significant advancement in understanding how microRNAs act as regulatory elements during Purkinje cell differentiation and maturation. The research not only sheds light on the developmental mechanisms governing these neurons but also raises broader implications for aging, brain plasticity, and neurological disorders.

“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,” explains senior author Giordano Lippi, associate professor of neuroscience at Scripps Research.

Previous studies had already suggested that microRNAs play a vital role in brain development, but their exact contribution to neuronal differentiation the process by which neural progenitors develop into specialized cell types 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.”

Purkinje cells are relatively rare in the brain, comprising less than 1% of the total cellular population in the cerebellum. However, their importance far outweighs their numbers. These neurons act as central hubs for processing and relaying information, receiving inputs from various parts of the brain and modulating motor control through extensive synaptic networks. Morphologically, they are among the largest neurons in the brain, with an elaborate tree-like structure composed of a single axon (the output signal pathway) and highly branched dendritic arbors (input structures). This complex architecture is crucial for their function, allowing them to form precise connections with climbing fibers, specialized input pathways that carry signals from other regions of the brain.

To better understand how microRNAs influence Purkinje cell development, the Scripps Research team devised a novel approach that allowed them to selectively inhibit microRNA function at specific developmental time points. Their experiments revealed that microRNAs are essential at two distinct phases of Purkinje cell maturation. When microRNA function was disrupted during the first week after birth, the Purkinje cells exhibited significantly less complex dendritic arbors and the cerebellum itself was smaller than normal. Conversely, when microRNA activity was inhibited during the third week of development, the Purkinje cells failed to form proper synaptic connections with climbing fibers. These results suggest that microRNAs serve a dual function: initially promoting structural complexity and later ensuring appropriate connectivity.

In collaboration with Ian MacRae, professor of integrative structural and computational biology at Scripps Research, the team also developed a sophisticated mouse model to determine which genes microRNAs were targeting. Using this system, they identified two critical microRNAs miR-206 and miR-133 as key regulators of Purkinje cell development. Additionally, they pinpointed four gene targets Shank3, Prag1, Vash1, and En2 that are directly influenced by these microRNAs. Notably, the researchers found stark differences when comparing the microRNA-target networks of Purkinje cells with those of pyramidal neurons, another major neuronal subtype. This indicated that Purkinje cells follow a unique microRNA blueprint during development.

For the first time, we were able to see that certain microRNAs are enriched in Purkinje cells but not in pyramidal neurons, Lippi notes. “By turning these microRNAs off, we demonstrated their essential role in shaping Purkinje cells' distinctive morphological features.

An interesting discovery in this study was the identification of three genetic targets that function as molecular "brakes" on cell growth. Under normal conditions, when microRNAs bind to these targets, the brakes are released, allowing Purkinje cells to develop their large and intricate dendritic structures. This finely tuned regulatory mechanism ensures that neurons achieve their proper form and function within the developing brain.

Importantly, several of these gene targets have previously been 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 to 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 yet, but this is something that we are looking forward to investigating in the future.

Looking ahead, the researchers plan to leverage their innovative toolset to further investigate microRNAs' roles in brain development, plasticity, and aging. Given that microRNAs are also implicated in various neurological conditions, from neurodevelopmental disorders to neurodegenerative diseases such as Alzheimer’s and Parkinson’s, this line of research holds promise for broader applications.

“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.”

As scientists continue to explore the molecular underpinnings of brain development, discoveries like this bring us one step closer to unraveling the complexities of neurodevelopmental disorders and identifying potential therapeutic targets. By deepening our understanding of how microRNAs orchestrate the intricate dance of neuronal differentiation and connectivity, researchers are paving the way for future breakthroughs in neuroscience, precision medicine, and regenerative therapies.

Source: https://www.scripps.edu/news-and-events/press-room/2025/20250402-lippi-neuron.html