Researchers at Northwestern University and the University of Pittsburgh have developed a new approach for identifying proteins inside different types of neurons in the brain of a living animal. The new mouse study offers insights into the brain’s distinct proteins and may lead to new therapeutic strategies against Parkinson’s disease and Alzheimer’s disease.

Their study is published in the journal Nature Communications in a paper titled, “Cell-type and subcellular compartment-specific APEX2 proximity labeling reveals activity-dependent nuclear proteome dynamics in the striatum.”

“The vertebrate brain consists of diverse neuronal types, classified by distinct anatomy and function, along with divergent transcriptomes and proteomes,” wrote the researchers. “Defining the cell-type specific neuroproteomes is important for understanding the development and functional organization of neural circuits. This task remains challenging in complex tissue, due to suboptimal protein isolation techniques that often result in loss of cell-type specific information and incomplete capture of subcellular compartments. Here, we develop a genetically targeted proximity labeling approach to identify cell-type specific subcellular proteomes in the mouse brain, confirmed by imaging, electron microscopy, and mass spectrometry.”

The researchers designed a virus to send an enzyme to a precise location in the brain of a living mouse. Derived from soybeans, the enzyme genetically tags its neighboring proteins in a predetermined location. After validating the technique by imaging the brain with fluorescence and electron microscopy, the researchers found their technique took a snapshot of the entire set of proteins (or proteome) inside living neurons, which can then be analyzed postmortem with mass spectroscopy.

“Similar work has been done before in cellular cultures. But cells in a dish do not 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 Northwestern’s Yevgenia Kozorovitskiy, PhD, associate professor and senior author of the study. “It’s a lot more challenging to do this work in the complex tissue of a mouse brain. Now we can take that proteomics prowess and put it into more realistic neural circuits with excellent genetic traction.”

By chemically tagging proteins and their neighbors, researchers can now see how proteins work within a specific, controlled area and how they work with one another in a proteome. Along with the virus carrying the soybean enzyme, the researchers also used their virus to carry a separate green fluorescent protein.

“The virus essentially acts as a message that we deliver,” Kozorovitskiy said. “In this case, the message carried this special soybean enzyme. Then, in a separate message, we sent the green fluorescent protein to show us which neurons were tagged. If the neurons are green, then we know the soybean enzyme was expressed in those neurons.”

Researchers have to divide proteins into peptides and then put them back together, which can take time and does not always guarantee a perfect process.

“We have been able to gain a lot of traction with genetic and RNA sequencing, but proteins have been out of the loop,” Kozorovitskiy said. “Yet everyone recognizes the importance of proteins. Proteins are the ultimate effectors in our cells. Understanding where proteins are, how they work, and how they work relative to each other is really important.”

“Mass spectroscopy-based proteomics is a powerful technique,” said Vasin Dumrongprechachan, a PhD candidate in Kozorovitskiy’s laboratory and the paper’s first author. “With our approach, we can start mapping the proteome of various brain circuits with high precision and specificity. We can even quantify them to see how many proteins are present in different parts of neurons and the brain.”

The researchers are looking forward to applying their approach to mouse models to better understand neurological illnesses.

“We are hoping to extend this approach to start identifying the biochemical modifications on neuronal proteins that occur during specific patterns of brain activity or with changes induced by neuroactive drugs to facilitate clinical advances,” Dumrongprechachan said.

“We look forward to taking this to models related to brain diseases and connect those studies to postmortem proteomics work in the human brain,” Kozorovitskiy said. “It’s ready to be applied to those models, and we can’t wait to get started.”

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