For the first time, researchers have developed a successful method to identify proteins in different types of neurons in the brains of living animals.
Led by Northwestern University & University of Pittsburgh, the new study represents a huge step forward in understanding the millions of different proteins in the brain. As the building blocks of all cells, including neurons, proteins are key to understanding complex brain diseases such as Parkinson’s and Alzheimer’s, which can lead to the development of new treatments.
The research will be published in the journal Nature Communications on August 11.
In the new study, the researchers developed a virus to deliver an enzyme to a precise location in the brain of a living mouse. The enzyme obtained from soybeans genetically marks its neighboring proteins at a predetermined location. After validating the technique by imaging the brain with fluorescence and electron microscopy, the researchers found that their technique took a snapshot of the entire set of proteins (or proteomes) in living neurons, which can then be analyzed post mortem with mass spectroscopy.
“Similar work has been done in cellular-culture before, but cells in a dish don’t work the same way they do in a brain, and they don’t have the same proteins in the same places doing the same things,” said Yevgenia Kozorovitskiy of the northwest., lead author of the study. “It’s much more difficult to do this work in the complex tissue of a mouse brain. Now we can take these proteomic prowess & put into more realistic neural circuits with excellent genetic traction.”
By chemically labeling proteins & their neighbors, researchers can now see how proteins function in a specific, controlled area and how they function together with one-another in a proteome. Along with the virus that carries the soybean enzyme, the researchers also used their virus to transport a separate green fluorescent protein.
“The virus is essentially acting like a message that we send,” Kozorovitskiy said. “In this case, the message carried this special enzyme. Then we sent the green fluorescent protein in a separate message to show us which neurons were marked.” If the neurons are green, we know that the soybean enzyme was expressed in those neurons.
Kozorovitskiy is the Soretta & Henry Shapiro Research Professor of Molecular Biology, an associate professor of neurobiology in Northwestern’s Weinberg College of Arts and Sciences & a member of the Chemistry of Life Processes Institute. She coordinated with Matthew MacDonald, assistant professor of psychiatry at the University of Pittsburgh Medical Center.
Protein Targeting Plays Catch-Up
While genetic targeting has completely changed biology & neuroscience, protein targeting is lagging behind. Researchers can amplify & sequence genes and RNA to identify their exact building blocks. However, proteins cannot be amplified and sequenced in the same way. Instead, researchers need to break down proteins into peptides and then put them back together, which is a slow & imperfect process.
“We got a lot of traction with genetic and RNA sequencing, but the proteins got out of the loop,” Kozorovitskiy said. “However, everyone recognizes the importance of proteins. Proteins are ultimate effectors in our cells. It is important to understand the location of proteins, how they work, and the interactions between them.
“Mass spectroscopy-based proteomics is a powerful technique,” said Vasin Dumrongsprechachan, a PhD student in Kozorovitskiy’s laboratory and lead author of the work. “With our approach, we can begin to map the proteome of various brain circuits with high precision & specificity. We might even quantify it to see how many proteins are in different parts of neurons & the brain.
Next Step: Better Understanding Of Brain Diseases
Now that this new system has been validated and ready to work, researchers can apply it to mouse models to detect diseases and better understand neurological diseases.
“We hope to extend this approach to start identifying biochemical modifications in neuronal proteins that occur during specific patterns of brain activity or changes induced by neuroactive drugs to enable clinical advances,” said Dumrongsprechachan.
“We hope to incorporate it into models related to brain diseases and link this research to postmortem proteomics in the human brain,” Kozorovitzki said. “It is ready for these models, and we can’t wait to get started.”
The research has been published in the Journal Of Nature Communication.