#brain diseases
(Image caption: Protein expression in the mouse brain for mass spectrometry analysis, visualized by fluorescence microscopy. Blue shows the outline of the brain. Green and magenta are selectively tagged proteins)
New technique identifies proteins in the living brain
For the first time, researchers have developed a successful approach for identifying proteins inside different types of neurons in the brain of a living animal.
Led by Northwestern University and the University of Pittsburgh, the new study offers a giant step toward understanding the brain’s millions of distinct proteins. As the building blocks of all cells including neurons, proteins hold the keys to better understanding complex brain diseases such as Parkinson’s and Alzheimer’s, which can lead to the development of new treatments.
The study was published in the journal Nature Communications.
In the new study, 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, 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.”
Kozorovitskiy is the Soretta and Henry Shapiro Research Professor of Molecular Biology, an associate professor of neurobiology in Northwestern’s Weinberg College of Arts and Sciencesand a member of the Chemistry of Life Processes Institute. She co-led the work with Matthew MacDonald, an assistant professor of psychiatry at the University of Pittsburgh Medical Center.
Protein targeting plays catch-up
While genetic targeting has completely transformed biology and neuroscience, protein targeting has woefully lagged behind. Researchers can amplify and sequence genes and RNA to identify their exact building blocks. Proteins, however, cannot be amplified and sequenced in the same manner. Instead, researchers have to divide proteins into peptides and then put them back together, which is a slow and imperfect 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 Ph.D. 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.”
Next step: Better understanding brain diseases
Now that this new system has been validated and is ready to go, the researchers can apply it to mouse models for disease 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.”
Why people on immunosuppressant drugs for autoimmune conditions have a higher incidence of an often-fatal brain disease may be linked to a mutation in a common virus, according to researchers at Penn State College of Medicine.
Progressive multifocal leukoencephalopathy (PML) is a rare disease of the brain’s white matter caused by the John Cunningham polyomavirus (JCV), a usually harmless virus that infects up to 80 percent of healthy adults.
In the past, the virus usually only developed into brain disease in individuals with suppressed immune systems, such as AIDS patients and organ transplant recipients on immunosuppressant medications. Over the past decade, however, new drugs for a variety of autoimmune conditions have been identified as a key risk factor for the brain disease.
The disease is now most common in multiple sclerosis patients who have the virus and who receive an immunosuppressant therapy called natalizumab. Patients with Crohn’s disease, psoriasis, rheumatoid arthritis, B cell lymphoma and chronic lymphocytic leukemia have also developed the devastating brain disease after immunosuppressant therapy with natalizumab and other drugs.
“Nobody comes away unscathed from PML—you either die or you’re left with a lifelong searing neurological defect,” said Aron E. Lukacher, chair and professor of microbiology and immunology. “Because we don’t know how the drugs cause the JC virus to amplify from a silent infection, we really have no way of controlling it.”
When other researchers studied the virus in patients with PML, they found a mutation in its protein shell, the part that allows it to bind to and infect human cells.
In the new study, Lukacher’s team sought to answer the question: Could this mutation affect how the immune system responds to the infection?
To answer this, the researchers developed a new mouse model of polyomavirus infection that shares many characteristics that are seen in progressive multifocal leukoencephalopathy.
They learned that mice with mutated polyomavirus strains had a reduced T cell response, the major part of the immune system that protects against the virus.
“We found that mouse polyomaviruses with a single amino acid change in their shell elicit a very different magnitude and quality of the T cell response that is needed to control the infection,” Lukacher said.
The researchers also found that a signaling protein called type I interferon controlled the difference in T cell response in mice with this mutant polyomavirus.
Type I interferon directly controlled the ability of the virus to grow in mouse cells, the researchers said. It also controlled the size of the T cell expansion and the function of these immune cells against the infection.
These findings raise the possibility that viral shell mutations may influence the T cell response to John Cunningham polyomavirus in humans, and play a role in the development of progressive multifocal leukoencephalopathy, the researchers wrote in the Journal of Virology.
It is still unknown how immunosuppressant therapies may cause these mutations in JCV, the researchers said. However, understanding the importance of the T cell response could help researchers prevent the development of progressive multifocal leukoencephalopathy in autoimmune patients.
“We need to find ways to improve the T cell responses in patients on these therapies,” Lukacher said.
