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Zars, Milescu labs awarded NSF ‘Early Concept’ grant to build new protein switches for neurons

Aug. 12, 2015

image shows two men and a woman in a lab

Drs. Mirela Milescu, Lorin Milescu, and Troy Zars were awarded a $300,000 BRAIN EAGER grant from the National Science Foundation.

A new technology is heating up the neuroscience world.

Thermogenetics — the combination of regulated temperature and genetics — uses genetic engineering to deliver special temperature-activated proteins to specific neurons in brains of experimental animals. Then, researchers can apply a specific temperature to control these neurons, basically turning them on or off at will.

Now, three Mizzou neurobiologists have been awarded an Early Concept Grant for Exploratory Research (EAGER) from the National Science Foundation to expand this technology. The technology could lead to a better understanding of brain disorders in humans.

The researchers, Troy Zars, Mirela Milescu, and Lorin Milescu, are faculty members of the university’s Division of Biological Sciences and Interdisciplinary Neuroscience Program.

“Thermogenetics is expanding the horizons of brain research by allowing us to precisely control specific neurons in the brain and measure behavioral changes,” says Zars, who is principal investigator of the grant. “So far, there are a relatively small number of proteins that do respond to temperature in way that is useful for work in flies. Our goal is to identify more of these special proteins, so that the technology can be used in other organisms.”

The proteins currently used for thermogenetic studies, called TRPs, are activated at about 28 degrees Celsius (82 degrees Fahrenheit). This activation temperature is fine for fruit flies, which as cold-blooded animals take on the ambient temperature of their environment.

“But in mouse, these proteins don’t work well because they’re always going to be activated, because the mouse is warm,” explains Zars, who has been studying temperature-response behaviors in fruit flies since 2000.

The solution is to find — or make — additional proteins that respond to a more useful range of temperatures. This team is doing both.

In one series of experiments, the scientists will comb through a large family of genes, called gustatory receptors, in search of a natural source of these special temperature-activated proteins. They will then express the genes in the neurons of flies to test their response to specific temperatures. Most relevant to the scientists will be identifying genes with different temperature properties.

The scientists have good reason to expect this gene family to harbor an abundance of these special proteins. Firstly, the family is already known to contain at least two genes that respond to a range of physiological temperatures. And, secondly, it’s a big family.

“There are about 68 genes in this family, so in terms of genes, there are many more genes than the TRPs,” says Mirela Milescu, who primarily focuses on the molecular biophysics of ion channels. “Why would there be so many if they don’t have fine response properties? This gives us the idea that maybe they are a better choice [than TRPs].”

In parallel experiments, the researchers will take a DIY approach and make their own temperature-activated proteins. Basically, their plan is to breakdown a receptor gene into its constituent parts, remove the thermosensor, and install it in another protein.

“Once we identify the building blocks of these particular proteins, we can create chimeric proteins by making mutations or switching parts of receptor proteins so that we can get an array of proteins that we can turn on or off at different temperatures,” says Mirela Milescu.

The natural and engineered proteins will be put into fly neurons to test the effects of changes in temperature on neural activity using advanced imaging techniques and software developed by Lorin Milescu.

“We have developed software tools that allow us to map neurons in the brain and associate their function with their position. With this software, we can construct spatial and temporal maps of the analyzed cells at multiple levels of resolution in real-time,” says Lorin Milescu. “Basically, we’ll end up with this map that is not just a picture of the neurons, but a functional map.”

While tested in flies, the new proteins will be useful in different organisms.

image of a fly brain with specific types of neurons highlighted

The natural and engineered proteins will be tested initially on select neurons of fly brains (as shown). However, the researchers expect to identify protein switches that are responsive to a range of temperatures and applicable across organisms.

“You start with Drosophila, but you can transfer them to rats or other types of research animals,” says Mirela Milescu. “Also, since each of us already works across multiple systems, we are well positioned to identify tools useful in multiple animal types.”

Zars echoes the importance of the team. “That’s why this team — the three of us working together — is really good. It’s a perfect combination of expertise, projects, and a big problem that can be useful for people, across organisms and across the country,” he says.

The thermogenetic technology builds on recent advances in neuroscience using light-sensitive proteins to control neural activity.

“By adding another type of protein on top of the light-responding proteins, we have a much better chance of looking at much more complex interactions,” says Zars.

EAGER grants support the federal BRAIN (Brain Research through Advancing Innovative Neurotechnologies) Initiative, a project that aims to map all the neurons in the brain. The two-year $300,000 awards support short-term, proof-of-concept projects.

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An abstract of the project, titled “BRAIN EAGER: Novel Thermo-genetic Tools for Extrinsic Control of Neuronal Circuits,” is available on the NSF Web site.

Written by: Melody Kroll

EurekAlert News Release

Related research strengths:
Molecular Biology, Neurobiology