An Outsider’s Perspective

Loren Looger thrives on collaboration. Since becoming a group leader at Janelia in 2006, he has lent his expertise in protein engineering to some 20 different lab groups on campus, as well as numerous colleagues across the globe. Most of his projects produce tools firmly rooted in neuroscience. For example, Looger has beefed up neural activity indicators, re-engineered a virus used to map nerve cell connections, and designed ion channels that switch neurons on and off. But Looger’s collaborations have also led him to other disciplines, such as clinical immunology, where he’s helping to deduce the molecular underpinnings of autoimmune disease.

Strengthening a Sensor

Although Looger has a catalog of more than a hundred projects, he’s probably best known for his collaborative work on a calcium sensor used to visualize neuronal activity.

When a nerve cell fires, calcium surges inside the cell. By tracking these calcium waves, scientists can watch neurons communicate with each other. Protein sensors that emit a fluorescent signal in the presence of calcium have been used in model organism studies since the late 1990s, but their signals were often weak and revealed little meaningful activity. So Looger set his sights on improving one of those sensors, GCaMP2.

First, he worked with Group Leader Eric Schreiter to solve the protein’s structure. Then, he iteratively tweaked the protein’s sequence to make it brighter and more responsive, sharing the products with any scientists game to try them. He found two willing partners at Janelia: Vivek Jayaraman, who tested the sensors in fruit flies, and Karel Svoboda, who did the same in mice. Cornelia Bargmann, an HHMI investigator at the Rockefeller University, rounded out the mix, testing the sensor in the worm Caenorhabditis elegans.

Of the numerous variants tested, the best by far was GCaMP3. Differing from its predecessor by just four amino acids, it was much more stable, better at binding calcium, and it fluoresced about three times as brightly as GCaMP2.

Neurons in the primary motor cortex that express the new genetically encoded calcium indicator GCaMP3 light up as they fire in sequence as the mouse moves a single whisker.

Looger’s team, working with the Genetically Encoded Neuronal Indicator and Effector (GENIE) Team Project at Janelia, has since improved on GCaMP3. The most recent iteration, GCaMP6, produces signals seven times stronger than past versions and is much more sensitive to brain activity in living animals. These sensors have been a boon to researchers worldwide who want to get a full picture of neuronal activity.

Teaching an Old Virus New Tricks

More recently, Looger teamed up with fellow Janelians Alla Karpova, Adam Hantman, and Joshua Dudman, as well as David Schaffer at the University of California, Berkeley, to create another tool that could have an enormous impact on neuroscience.

For scientists to fully understand brain function, they need to see how different types of neurons connect with each other. One tactic is to use a virus to deliver genes that code for fluorescent proteins to nerve cell nuclei; once there, the genes are expressed and the neurons glow, making them easy to visualize.

Currently, many researchers use the harmless adeno-associated virus (AAV) for this task. AAV can be taken up by axons and transported to the cell body, where its genetic payload is transcribed. Unfortunately, naturally evolved AAVs are very bad at retrograde transport, making the journey from axon to nucleus slow and often impossible. “This is problematic, because some axons (frequently the most interesting ones) can be really, really long,” says Looger.

By changing the protein shell of AAV, Looger and his collaborators increased its ability to get inside axons and move quickly to the cell body, improving its retrograde transport by more than 100 times over existing strains.

“We still don’t know exactly how the new virus works,” concedes Looger. “That’s the next step.” He has a strong suspicion that the virus is hitching a ride on a transport molecule inside the cell.

When injected into the mouse brain, the AAV that the team engineered is able to infect, and light up, neurons projecting to the different nuclei. This facilitates “projectomics” research in ways that were not possible with existing AAV viruses (which do not perform retrograde transport as part of their lifecycle in the wild).

Investigating Immune Disease

Although many of Looger’s tools address needs within the neuroscience community, other fields may benefit from them as well. For example, the re-engineered AAV could be used as a vector for gene therapy. “We’re not just working on neural imaging,” he says. “Our tools are being used to study diseases such as tuberculosis, malaria, diabetes, and cancer, to name a few.”

Looger’s work has also extended to lupus. “About five years ago this guy emailed me and said, ‘You don’t know me, but we have a mutual friend and he says you’re good with proteins,’” Looger recalls. That guy was Swapan Nath, a human geneticist at the Oklahoma Medical Research Foundation. Nath had analyzed genetic data from more than 19,000 people with lupus and found a handful of gene variants that appeared to increase the risk of lupus in African Americans. But he wanted help nailing down what those gene changes did.

Within two hours of receiving the email, Looger had analyzed the data and sent Nath a hypothesis of what each variant was doing. The analysis suggested how the sequence changes might cause the cellular dysfunctions associated with lupus, by disrupting the function of the protein encoded by the gene or altering how the protein interacts with other proteins.

Looger and Nath are now working on their sixth and seventh papers together. Their latest collaboration focuses on lupus-associated mutations that occur in noncoding portions of the genome.

“That collaboration has been really fun and hugely productive too,” says Looger. “I could not be further from this field in terms of training, so I bring this complete outsider’s perspective.”

That outsider’s perspective is the key to success in many of Looger’s projects. Because he’s not entrenched in the dogma of his collaborators’ disciplines, Looger says the assumed limits of those fields rarely restrict his imagination. “I don’t know what’s impossible,” he says. “So we try a lot of things that people say will never work...and a lot of it has been successful.”