Axons are extremely thin membrane fibers that are specialized for carrying action potentials. While their diameter varies along their length, the factors that shape their structure remain unclear.
Recently, researchers from Johns Hopkins Medicine have provided new insights suggesting that the armlike formations of brain cells in mammals may have a different appearance than scientists have believed for over a hundred years. Their research on mouse brain cells indicates that these cells’ axons — the extensions that connect and communicate with other brain cells — are not the cylindrical tubes typically illustrated in textbooks and on the internet but rather resemble pearls strung together on a thread.
“Understanding the structure of axons is important for understanding brain cell signaling,” says Shigeki Watanabe, Ph.D., associate professor of cell biology and neuroscience at the Johns Hopkins University School of Medicine. “Axons are the cables that connect our brain tissue, enabling learning, memory and other functions.”
Scientists have made a groundbreaking discovery regarding axon beading—those intriguing pearl-like structures in axons that emerge in dying brain cells and in patients with Parkinson’s and other neurodegenerative diseases. This beading phenomenon is a direct consequence of the loss of neuronal membrane and skeletal integrity, highlighting the fragility of our neural systems.
In a healthy state, axons are elegantly structured like tubes with a consistent diameter, decorated with bubble-like synaptic varicosities that play a crucial role in neurotransmitter storage, enabling vital communication between brain cells.
Watanabe first identified the phenomenon of recurrent axon pearling in the nervous systems of worms. His curiosity intensified after an enlightening discussion with Swiss scientist Graham Knott, Ph.D. They reviewed a pivotal 2012 study from Harvard University revealing repetitive “skeletal” components in axons. This spurred the researchers to design experiments targeting the axon skeleton to determine whether its removal would eliminate the pearled structures.
Jacqueline Griswold, a dedicated Johns Hopkins graduate student, and the study’s first author, explored this idea but was unable to observe any changes in axon pearling. Eager to delve deeper, Watanabe and Griswold partnered with Padmini Rangamani, Ph.D., a theoretical biophysics expert at the University of California San Diego School of Medicine, to investigate the axons’ physical properties with greater precision.
To visualize these tiny axons, which are astonishingly 100 times smaller than the width of a human hair, the team employed high-pressure freezing electron microscopy. This advanced technique, similar to traditional electron microscopy but optimized for preserving cellular structure, allowed them to freeze mouse neurons, ensuring that the delicate shapes of these crucial structures were meticulously maintained for analysis.
“To see nanoscale structures with standard electron microscopy, we fix and dehydrate the tissues, but freezing them retains their shape — similar to freezing a grape rather than dehydrating it into a raisin,” says Watanabe.
The researchers investigated three categories of mouse neurons: those cultivated in a laboratory, those procured from adult mice, and those sourced from mouse embryos. The neurons lacked myelination, meaning they did not possess the myelin sheath that usually encases the axon.
Among the numerous images captured from the tissue samples, the researchers observed the distinctive bubbly, pear shape of the axons. The scientists referred to the pearl-like formations where the axon expands as “non-synaptic varicosities.”
“These findings challenge a century of understanding about axon structure,” says Watanabe.
The researchers also employed mathematical modeling to investigate whether the composition of the axon membrane affected the form or existence of the pearl-like structures. They discovered that straightforward mechanical models could effectively account for these formations.
Moreover, tests using the mathematical model alongside mouse brain samples indicated that raising the concentration of sugars in the solution surrounding the axon or diminishing the tension in the axonal membranes led to a decrease in the size of the pearl structures.
In a separate experiment, the researchers extracted cholesterol from the neuron’s membrane to enhance its fluidity and reduce stiffness. Under these circumstances, they observed a decrease in pearling in both the mathematical models and the mouse neurons, as well as a diminished capacity of the axon to convey electrical signals.
“A wider space in the axons allows ions [chemical particles] to pass through more quickly and avoid traffic jams,” says Watanabe.
The researchers have harnessed high frequency electrical stimulation on mouse neurons, resulting in significant swelling of the pearled structures along the axons—by an impressive 8% in length and 17% in width—lasting for at least 30 minutes post-stimulation. This enhancement notably accelerated the speed of electrical signals.
However, an intriguing finding occurred when cholesterol was eliminated from the membrane; the axon’s pearls reverted to their original state, with no impact on the speed of electrical signals. In a critical next step, the research team aims to study axonal “arms” within human brain tissue obtained ethically from individuals undergoing brain surgery and from those who have succumbed to neurodegenerative diseases.
Journal reference:
- Jacqueline M. Griswold, Mayte Bonilla-Quintana, Renee Pepper, Christopher T. Lee, Sumana Raychaudhuri, Siyi Ma, Quan Gan, Sarah Syed, Cuncheng Zhu, Miriam Bell, Mitsuo Suga, Yuuki Yamaguchi, Ronan Chéreau, U. Valentin Nägerl, Graham Knott, Padmini Rangamani & Shigeki Watanabe. Membrane mechanics dictate axonal pearls-on-a-string morphology and function. Nature Neuroscience, 2024; DOI: 10.1038/s41593-024-01813-1