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Quantitative Proteomics Reveals New Gene Networks in Nerve Regeneration


NEW YORK (GenomeWeb) – Scientists from Boston Children's Hospital have used quantitative proteomics to uncover new roles for gene networks and molecular pathways in nerve regeneration. The technique, especially when used in conjunction with deep sequencing, is providing neuroscientists with more data on such gene networks, and with ever more sensitive instruments, is poised to make big contributions to the field of neurobiology.

The scientists, led by Zhigang He and Judith Steen, found some of the usual suspects responsible for the regeneration of retinal ganglia cells in mice, such as the cell growth regulator mTOR, but also implicated new gene networks in neuron regeneration, such as c-Myc, a proto-oncogene. They published their study last week in Neuron.

Though deep sequencing offers a way for scientists to see genes involved in neuronal processes, combining it with proteomics has become an essential tool for neuroscientists, according to Steen. "By measuring proteins, you get a more direct, downstream readout of the system," she said, noting that the technique can be used to great effect throughout neuroscience. "Proteomics is helpful in Alzheimer's to understand the nature of disease and to understand systems-level pathways that lead to disease or neurodegeneration as a whole."

Proteomics technology is advancing rapidly and the instrumentation is already much more sensitive than when the scientists collected their data for the neuron regeneration study, which was four years ago.

The Steen and He labs were trying to answer a fundamental question about why injured nerves in the central nervous system lose the ability to regenerate, for example, after traumatic brain injury and spinal cord injury. For many cells in the central nervous system, regeneration only occurs after injury; however, often as a result of traumatic brain injury, damage to the long fibrous projections of the neuron known as axons cannot regenerate, He said.

A previous study from He's lab showed that one of the pathways involved in axon regeneration is the mTOR pathway, which is regulated by a gene called PTEN. "If we delete PTEN and activate mTOR, that allows axons to regenerate," He said.

The new study's proteomic analysis of injured and non-injured retinal ganglia cells (RGCs) showed that both mTOR and the gene c-Myc were downregulated in the injured cells. Measuring c-Myc expression levels with qPCR showed that expression decreased by 70 percent after injury, they wrote.

The mTOR pathway was expected to be involved, but c-Myc, a proto-oncogene, had not been associated with axon regeneration before. Follow-up studies showed that overexpressing c-Myc could promote axon regeneration in injured mice.

"It seems like both pathways are important for cell metabolism, especially anabolic metabolism. Cells need to use these two different pathways to synthesize proteins and lipids for dividing cells," He said. "They need to switch the metabolism program and allow neurons to synthesize new proteins and lipids essential for axon regeneration."

Though the pathways are not ideal therapeutic targets, Steen and He said the study hints at manipulating cell metabolism as a strategy for regenerating axons.

It was the analysis of proteins found in injured cells versus uninjured cells that allowed the scientists to find the role c-Myc played in neuron regeneration.

"With state of the art instrumentation, we can sequence and quantify many, many more proteins and even quantify it in a context where we have limited sample material," Steen said. "We were looking at only a very small subset of neurons from retina, which make up less than 5 percent of the total number of neurons in the retina."

Proteomics allowed them to look specifically at the RGCs in live tissue, both injured and healthy. "I think our paper was the first to study a subset of neurons," Steen said. "That has never been done before to my knowledge."

There are many types of neurons in the brain, but in many neurological diseases only a particular type of neuron is affected. "So the analyte change in a particular neuronal subtype will be very crucial" to understanding those diseases, He said.

"You could use deep sequencing methods and look at the RNA levels. We decided to look at proteins because of fact that proteins are the machinery that actually do work in the cell," Steen said. "Not all genes that are transcribed are translated into proteins. The most proximal readout of injuries is the proteomics."

The proteomics in the study was done with a Thermo Fisher Scientific Orbitrap Classic instrument, though Steen noted that the technology has improved greatly from when the data were taken four years ago.

Informatics tools were also important to revealing c-Myc's role in axon regeneration. The scientists needed to uncover the networks of genes affected by injury, which was done using software built in house over seven years, Steen said, in conjunction with other available pathway analysis tools. Steen said the software is open source, available in Bitbucket, a software sharing service, or from her lab's website.

The hope is that with the tools available and the benefits of proteomics demonstrated, others in the field of neuroscience will use the technique more.

"There are not many labs that actually do proteomics in neuroscience, but I believe it will become more widespread," she said.