NEW YORK (GenomeWeb News) – A large-scale proteomic study is helping researchers unravel the pathways influencing neuron signaling and function.
Scientists at the University of California, San Diego, in conjunction with the Pacific Northwest National Lab in Richland, Wash., used global proteome profiling to investigate protein networks in neuroblastoma cells. Using quantitative mass spectrometry, they identified 4,855 proteins in cells undergoing neuritogenesis, a process involved in brain development, and mapped these into distinct and overlapping networks. Their findings appear online this week in the Proceedings of the National Academy of Sciences.
“It’s going to be a bit surprising, I think, to many people,” UCSD pathologist Richard Klemke told GenomeWeb Daily News.
Brain and nerve development rely on a neuron’s ability to grow and differentiate, and a process called neuritogenesis is behind this ability. During neuritogenesis, long, thin structures called neurites extend out of nerve cell bodies in response to external signals that guide their development. Eventually, these neurites differentiate into the cell axons and dendrites that stretch between neurons, relaying and receiving nerve signals.
It’s unclear, though, how cell signaling controls this process. Identifying the proteins within neurites is one way to begin evaluating which networks govern neuritogenesis. But in the past, scientists have had problems separating neurites from the cell body, hindering proteomic analysis.
To address this, Klemke and his team developed a new neurite purification technique in neuroblastoma cells, a type of nerve tissue cancer. They prompted neurites to grow through a filter and then separated them from the cell body by scraping them into a mass spec buffer. They then quantified the relative protein abundance in both the neurites and the cell body using large-scale, two-dimensional liquid chromatography-coupled tandem mass spectrometry.
“This is really the first sort of large-scale analysis of these small [neurite] structures,” Klemke said.
Of the 4,855 proteins they identified, less than half of these — 1,960 — were equally distributed between neurites and soma. 1,679 of the remaining proteins were enriched in the cell body, while the remaining 1,229 were found to a greater degree in the neurite projections.
Using bioinformatics tools such as Babelomics’ online gene ontology resource and Ingenuity's pathway analysis software and database, they were able to identify the most statistically significant protein networks.
Their results suggest the neurite houses some 39 different protein pathways, including those controlling the actin cytoskeleton, the framework cells use to grow and extend. The cell body, on the other hand, contains at least seven pathways, involved in everything from a cell’s response to estrogen to its entry into the cell cycle. Klemke and his colleagues identified an additional five pathways that were present in both the neurite and cell body.
The team also used this approach to hone in on a cellular pathway containing the proteins Cdc42 and Rac, also involved in cytoskeletal growth. Their subsequent functional work suggests several guanine-nucleotide exchange factors and GTPase-activating proteins regulate these pathways, depending on the stage of neurite development.
“It is a complex network of proteins,” Klemke said, “There’s not a single protein that’s key. They all work together.”
In the future, the group hopes to use a similar proteomic approach in primary neuron cells. They also plan to apply the technology to determining which proteins are regulated by phosphorylation. Such a phosphoproteome map may be key to understanding network regulation. “One can then build a signaling network from all this information,” Klemke said.
And, he added, being able to understand — and perhaps control — neuron signaling may make it possible to “rewire” the brain or nervous system and lead to new treatments for neurodegenerative diseases such as Alzheimer’s or spinal cord injuries. “We want to be able to tackle important human disease,” Klemke said.