Scientists say protein separates message-bearing ‘bubbles’ at intersection between brain cells, deepening understanding of cognition

Researchers at Johns Hopkins Medicine say they unexpectedly found new information about a protein’s special role in getting brain cells to communicate at the right time and place in experiments with genetically engineered mice.

The finding about the protein intersectin, they say, advances scientific understanding of a key process in how the mammalian brain forms memories and learns, and may help advance treatments for cognitive disorders including Down syndrome, Alzheimer’s disease and Huntington’s disease.

A report of the new findings, funded in part by the National Institutes of Health, was published July 8 in the journal Nature Neuroscience. 

Specifically, the researchers found that intersectin keeps tiny, message-carrying bubbles inside brain cells in a particular location until they are ready to be released to activate a neighboring brain cell. The protein does so by creating a physical boundary between these bubbles, similar to how oil separates from water.

Message transfer from brain cell to brain cell is key to information processing, learning and forming memories. The bubbles, synaptic vesicles, are housed within the synapse — the connection point where brain cells communicate. In typical synapses within the brains of mammals, 300 synaptic vesicles are clustered together in the intersection between any two brain cells, but only a few of these vesicles are used for such message transfer, researchers say. Pinpointing how a synapse knows which vesicles to use has long been a target of research by those who study the biology and chemistry of thought.

“We found that these tiny bubbles have a distinct domain where they want to be,” says Shigeki Watanabe, Ph.D., associate professor of cell biology at Johns Hopkins Medicine, who led the research. “Keeping them at particular locations within a synapse enables the brain to decide how and when to use them while thinking and processing information.”

In an effort to better understand the operation of these synaptic vesicles, Watanabe and his team designed a study that first focused on endocytosis, a process in which brain cells recycle synaptic vesicles after they are used for neuronal communication.

Already aware of intersectin’s general role in endocytosis and neuronal communication, the scientists genetically engineered mice to lack the gene that codes for intersectin. However, and somewhat to their surprise, Watanabe says removing the protein did not appear to halt endocytosis in brain cells.

The research team refocused their experiments, taking a closer look at the synaptic vesicles themselves.

Using a high-resolution fluorescence microscope to observe where intersectin is in a synapse, the researchers found it in between vesicles that are used for neuronal communication and those that are not, as if they are physically separating the two.

To further understand the role of intersectin at this location, they used an electron microscope to visualize synaptic vesicles in action across one billionth of a meter. In all the nerve cells from mice lacking this protein, the scientists say synaptic vesicles close to the membrane were absent from the release zone of the synapse, the place where the bubbles would discharge to nearby neurons.

“This suggested that intersectin regulates release, rather than recycling, of these vesicles at this location of the synapse,” says Watanabe.

Using a technique called zap and freeze microscopy, the scientists stimulated neurons in the brains of mice to capture the movement of synaptic vesicles on a millisecond timescale and at a nanometer resolution.

In normal mice, the scientists saw vesicles fusing with the brain cell membrane within a millisecond after stimulation. Then, new synaptic vesicles came and filled the vacated release sites of the synapse within about 15 milliseconds.

In two genetically engineered lines of mice, one lacking intersectin and another lacking the endophilin protein, which binds to intersectin, new vesicles could not be recruited to the vacated release sites. Similarly, vesicles within nerve cells of mice with mutations that blocked the interaction of these two proteins also slowed the local replenishment of synaptic vesicles that carry information from neuron to neuron.

“When information is processed in the brain, this replenishment process needs to happen in just a few milliseconds,” says Watanabe. “When you don’t have vesicles staged and ready to go at the release sites or the active zones, then neurotransmission cannot continue.”  

In future research, the scientists say they aim to better understand how intersectin shuttles new synaptic vesicles to release sites.

Funding support for this research was provided by the National Institutes of Health (GM118177, FA95501610052, R01GM136711, S10 OD016374, P50CA098252), the Air Force Research Laboratory, the Defense Advanced Research Projects Agency, the Sol Goldman Pancreatic Cancer Research Center, a Johns Hopkins Discovery Award, the W.W. Smith Charitable Trust Award, the National Science Foundation, the American Heart Association, a Johns Hopkins Albstein Research Scholarship, the Wellcome Trust, the John Fell Fund, La Caixa Foundations, EU-Horizon 2020 MIA-Portugal, Deutsche Forschungsgemeinschaft; the German Center for Neurodegenerative Diseases, the European Research Council, the Human Frontiers Science Organization, the German Dementia Association, and the Guangzhou Elite Project.

Additional researchers who conducted the study are Tyler Ogunmowo, Chintan Patel, Renee Pepper, Annie Ho, Sumana Raychaudhuri and Brady Maher of Johns Hopkins; Christian Hoffmann, Han Wangfrom and Dragomir Milovanovic from the German Center for Neurodegenerative Diseases, Sindhuja Gowrisankaran and Johanna Idel from the European Neuroscience Institute; Benjamin Cooper from the Max Planck Institute for Multidisciplinary Sciences and Ira Milosevic from the University of Oxford.

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