A pop-up repair shop for damaged neurons

Neurons, the building blocks of the brain and nervous system, use biological wires (axons and dendrites) to send signals to each other. Neurons can also rewire their connections as the nervous system learns and remembers. Most impressively, some neurons can even repair their far-flung axons, which can be deployed as far as three feet from the rest of the cell.

Somehow, after an injury, axons are able to produce the proteins required to repair themselves, far from the molecular machines in the cell body that are typically responsible for protein production. Thanks to a technique developed in the lab of Alma Burlingame, PhD, scientists have now solved a big part of this long-distance mystery, showing that a particular genetic message, stored along each axon, rapidly sounds the alarm during an injury and kick-starts the synthesis, or translation, of new proteins.

The research holds promise for someday harnessing the nervous system’s own repair programs to develop therapies for neurodegeneration and traumatic brain injury.

“When an axon is damaged, RNA stored in the axon is quickly translated into a protein called mTOR, which then translates a bunch of other proteins that are necessary to alert the rest of the neuron of the injury,” said Burlingame, a faculty member in the UCSF School of Pharmacy’s Department of Pharmaceutical Chemistry and the director of the UCSF Mass Spectrometry Facility (MSF).

The work, a collaborative effort led by Mike Fainzilber, PhD, at the Weizmann Institute of Science in Rehovot, Israel, was published March 23 in Science: Locally translated mTOR controls axonal local translation in nerve injury. This study used the Burlingame Lab’s recently unveiled method for rapidly monitoring protein synthesis, which itself was published on March 6 in PNAS: Revealing nascent proteomics in signaling pathways and cell differentiation.

A method born in the right place, at the right time

The discovery was the result of an ongoing collaboration between Burlingame’s lab, which uses mass spectrometry to identify many different proteins at once, and the labs of Fainzilber and Jeff Twiss, PhD (University of South Carolina), which study how neurons grow and repair themselves. And it was based on a method that the Burlingame lab had been developing in collaboration with Craig Forester, MD, PhD, in the lab of Davide Ruggero, PhD (UCSF).

Qian Zhao, PhD, a post-doctoral researcher at the UCSF MSF and second author on the PNAS paper, had been working with a modified version of a commonly available molecule, puromycin. When introduced to a cell, puromycin attaches itself to growing proteins. Zhao used a particular version of puromycin, O-propargyl-puromycin (OPP), which allowed her to actually pull newborn proteins out of a cell.

After using OPP to isolate growing proteins, Burlingame, Zhao, and PNAS paper first author Craig Forester could then use a technique called mass spectrometry—Burlingame’s specialty—to identify which proteins the cell was in the midst of churning out.

Meanwhile, Fainzilber’s team was investigating the particulars of local translation of proteins in damaged neurons, and Burlingame suspected his lab’s new strategy might prove useful for Fainzilber’s work.

“By chance, we had just developed this translation strategy using OPP,” said Burlingame. “It was still in its early stages, and we had just been able to show that the method was working. [Fainzilber’s group] then did the experiments that actually showed which proteins were being produced in these damaged axons.”

Remarkably, the method “worked really well” for Fainzilber’s study, said Burlingame. The group was able to observe local translation of mTOR and other proteins just two to three hours after injury to axons, when proteins produced in the cell body would have taken up to 12 hours to travel to the injury site.

Better biology through chemistry

mTOR is famous for being a “master regulator” of many other genes, and dysfunctional mTOR is associated with diseases as diverse as cancer, diabetes, and neurodegeneration. These findings may soon inspire new therapies for such conditions.

Burlingame is excited by the prospect of other labs applying his group’s technique for quickly capturing and identifying newborn proteins using OPP. Mass spectrometry is now used frequently to study many proteins simultaneously, but OPP—a molecule developed by chemists for biologists—holds great promise for scientists seeking to understand rapid changes in translation, like those that occur right after an injury.

“Our findings really demonstrate UCSF’s emphasis on collaboration,” said Burlingame. “Chemistry has a lot to offer to biology, and these advances are only possible when different fields come together.”

Additional authors on the PNAS study include Nancy J. Phillips, Anatoly Urisman, Robert J. Chalkley, Juan A. Oses-Prieto, and Li Zhang. Funding was provided by the Campini Foundation, the Leukemia and Lymphoma Foundation, the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, the Howard Hughes Medical Institute, and the National Institutes of Health.

Additional authors on the Science study include Marco Terenzio, Sandip Koley, Nitzan Samra, Ida Rishal, Pabitra K. Sahoo, Anatoly Urisman, Letizia Marvaldi, Juan A. Oses-Prieto, Cynthia Gomes, Ashley L. Kalinski, Agostina Di Pizio, Ella Doron-Mandel, Rotem Ben-Tov Perry, and Indrek Koppel. Funding was provided by the European Research Council, the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, the Minerva Foundation, the Israel Science Foundation, the Department of Defense, the National Institutes of Health, and the Company of Biologists.