Breakthrough Provides New Tool for Degenerative Disease Studies

http://oregonstate.edu/dept/ncs/news...uperoxide.html


CORVALLIS, Ore. – Scientists in the Linus Pauling Institute at Oregon State University have discovered a new technique to let them watch, visualize and precisely measure a key oxidant in animal cells, an important breakthrough that could dramatically speed research on everything from Lou Gehrig’s Disease to heart disease, hypertension, diabetes and aging.

The findings are being published online this week in Proceedings of the National Academy of Sciences, a professional journal. They could open the door to major advances on some of the world’s most significant degenerative diseases, researchers say.

The OSU scientists, in collaboration with Molecular Probes-Invitrogen of Eugene, Ore., found a chemical process to directly see and visualize “superoxide” in actual cells. This oxidant, which was first discovered 80 years ago, plays a key role in both normal biological processes and – when it accumulates to excess – the destruction or death of cells and various disease processes.

“In the past, our techniques for measuring or understanding superoxide were like blindly hitting a box with a hammer and waiting for a reaction,” said Joseph Beckman, a professor of biochemistry and director of the OSU Environmental Health Sciences Center. “Now we can really see and measure, in real time, what’s going on in a cell as we perform various experiments.”

In research on amyotrophic lateral sclerosis, or Lou Gehrig’s Disease, which is one of his lab’s areas of emphasis, Beckman said they have used the new technique to learn as much in the past three months about the basic cell processes as they did in the previous 15 years. Hundreds of experiments can now rapidly be done that previously would have taken much longer or been impossible.

“This will enable labs all over the world to significantly speed up their work on the basic causes and processes of many diseases, including ALS, arthritis, diabetes, Parkinson’s disease, Alzheimer’s disease, heart disease and others,” Beckman said. “And it should be especially useful in studying aging, particularly the theory that one cause of aging is mitochondrial decay.”

The process of oxidation in the body, researchers say, is one that’s fundamental to life but also prone to problems. Oxygen in the cells can be reduced to a molecule called superoxide, which is part of normal immune system processes and may also have other functions – it was first named by OSU alumnus Linus Pauling in 1934.

“Oxygen is actually one of the more toxic molecules in the environment,” Beckman said. “Breathing 100 percent pure oxygen will destroy your lungs in about three days because it increases the formation of superoxide.”

Superoxide is efficiently removed by an enzyme, superoxide dismutase. Antioxidants in food, such as vitamin C and E, are also part of this process. And in healthy animals, including humans, this delicate balancing act can work well and cause few problems. But sometimes the process breaks down and excess levels of superoxide begin to accumulate and lead to a wide variety of degenerative diseases.

Prior to this, there was no direct and accurate way to measure superoxide or its origin from the two places that produce it, the cell’s cytosol or mitochondria. Now there is.

With the new system developed at OSU, researchers can use a fluorescent microscope, a fairly standard laboratory tool, to actually see levels of superoxide and observe changes as experiments are performed with living cells.

“If we poison the mitochondria, using something like the pesticides that have been implicated in Parkinson’s disease, we can actually see superoxide levels begin to rapidly rise,” Beckman said. “You get a similar reaction if a growth factor is added that’s implicated in the development of Lou Gehrig’s Disease.”

The data available from this new technology, Beckman said, are so profound that for some time many in the science community didn’t believe it was possible.

“This will become a critical tool in learning how superoxide works in a cell,” he said. “I’ve been studying this for more than 10 years and never thought we would have such a clear and accurate picture of what’s going on inside a living cell.”

In their research on ALS, for instance, OSU scientists have used the new system to actually see cells eating themselves alive and dying from excess superoxide production. A new compound is in phase one clinical trials that appears to inhibit this process and may ultimately provide a therapy for the disease.

Oxidative stress resulting from mitochondrial dysfunction has already been implicated in neurodegeneration, aging, diabetes and cancer, the researchers said in their report. The new findings could rapidly speed research in all of those fields, they said.

This research was funded by grants from the National Institutes of Health and the OSU Environmental Health Sciences Center.

In a related study....

I'm posting the entire article because it's a press release:


Insight Into Parkinson's Disease May Be Offered By A Laser Probe Of A Brain Pigment's Anatomy
27 Sep 2006

In a finding that may offer clues about Parkinson's disease, a team led by Duke University researchers used a sophisticated laser system to gain evidence that a dark brown pigment that accumulates in people's brains consists of layers of two other pigments commonly found in hair.

Other scientists previously had determined via chemical analysis that the dark pigment, called neuromelanin, is composed of the two pigments: eumelanin, found in black-haired people, and pheomelanin, found in redheads. But how those pigments are arranged structurally remained unknown -- and this structuring may prove to be of critical importance, according to the researchers.

In addition, in 2005 a Duke team that included some of the same scientists involved in the current study reported using the laser system to establish that pheomelanin is chemically disposed to activate oxygen while eumelanin is not. Oxygen activation is suspected to play a role in the neurogenic cascade of events behind Parkinson's disease.

In the new report, investigators from Duke, North Carolina State University and the Institute of Biomedical Technologies in Segrate, Italy, outlined evidence that neuromelanins isolated from human brains have cores of oxygen-activating pheomelanin covered by a protective surface of eumelanin.

"This is the first piece of morphological data about how these pigments are constructed," said study leader John Simon, the George B. Geller Professor of chemistry at Duke.

The team published the findings online during the week of Sept. 25 in the journal Proceedings of the National Academy of Sciences. The research was funded by the U.S. Air Force Office of Scientific Research, through grants to the Duke University Free Electron Laser Laboratory, and by the Italian Fund for Basic Science.

The findings "should stimulate renewed interest in the roles of neuromelanin in the pathogenesis of Parkinson's disease, the second most prevalent neurodegenerative disorder," Shosuke Ito, a chemist at Japan's Fujita Health University School of Health Sciences, wrote in a companion commentary published in the journal.

According to the team's report, whose first author is Simon's graduate student, William Bush, neuromelanin granules begin appearing in human brains between ages 3 and 5, and their concentrations increase steadily thereafter.

However, neuromelanin levels drop precipitously in the brains of Parkinson's patients, who also experience a death of brain cells that are darkly pigmented and an increase in brain tissue concentrations of the metal iron.

Brain cells that produce dopamine, a key neurotransmitter disrupted in Parkinson's disease, experience high levels of oxidation as that dopamine is made, the researchers noted.

Scientists have hypothesized that brain cells synthesize neuromelanin to serve as a defense mechanism against high oxidation stress, the team's report said.

Neuromelanin's layered granular structure could help protect brain cells from damage in several ways, Ito wrote in his commentary.

Having eumelanin at their surfaces would protect the granules with a pigment known to efficiently bind iron and other molecules that could otherwise play a role in oxidative damage. If the underlying core of pheomelanin were instead positioned at the surface, "the neuro-protective role of neuromelanin would not be expected," Ito added.

However, eumelanin is limited in how much iron it can take up, and other scientists have proposed that iron over-saturation at the granules' surfaces could contribute to the high levels of the metal in the brains of Parkinson's victims.

"Increased oxidative stress under such conditions could result in degradation of the eumelanic surface of neuromelanin," Ito wrote. That could expose a pheomelanin core "that is not only ineffective in iron-binding, but also behaves as a pro-oxidant itself," he added.

"Once these neuromelanin granules start getting chewed into, an environment is created that is much more pro-oxidation," Simon said. "As pigment starts to get eroded, you can imagine how oxidative stress could be increased in multiple ways."

In the study, which Ito called "sophisticated," the researchers used a special laser device that makes light with electrons that have been freed from their usual bondage to atoms. Housed in a large bay in the Duke University Free Electron Laser Laboratory, the device can be "tuned" step-by-step to produce light at a variety of different wavelengths, with each wavelength probing different energy regions in target molecules.

The team also used a device called a photoelectron emission microscope to resolve individual neuromelanin granules and distinguish between the two pigment types.

Using these devices in combination, the researchers could pinpoint the "oxidation potentials" of molecules coating the surfaces of neuromelanin granules. Oxidation potentials measure how likely given chemicals are to activate oxygen by giving up electrons. Activated oxygen can produce compounds called radicals that can stress cells.

The team found that oxidation potentials of molecules at the surfaces approximated those found in black hair pigments in the 2005 study. "That meant it was eumelanin, which is pretty antioxidant," Simon said.

The laser beams could not penetrate beneath the granules' surfaces to record oxygen potentials nearer their cores. But previous chemical analyses by other researchers had established that neuromelanin is a mixture of both red and black hair pigments. So, the new finding suggests "a structural motif, with pheomelanin at the core and eumelanin at the surface," the team reported.

"Something special is happening, where the red pigment is getting encased in the black," Simon said. "So the red, being fairly pro-oxidant, is being encased in this antioxidant pigment."

Simon's group could only deduce the probable structure of neuromelanin, rather than measure it directly, because scientists have so far been unable to synthesize the pigment from chemical building blocks in a form that duplicates the natural version, he said.

"No one knew how to test or probe these things," Simon said. "And I can't overestimate how difficult it was to get materials to test." His group worked with small amounts of autopsied brain tissues provided by a research group led by Luigi Zecca at the Italian Institute of Biomedical Technologies.

Other researchers in the study were Glenn Edwards, director of the Duke University Free Electron Laser Laboratory; Robert Nemanich and Jacob Garguilo of N.C. State; and Fabio Zucca and Alberto Albertini of the Italian Institute of Biomedical Technologies.

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Contact: Monte Basgall
Duke University