ALBUQUERQUE, N.M., Oct. 15 -- Anyone visiting a nursing home has seen the horror of humans surviving beyond their brains' ability to make sense of their surroundings. That loss of discrimination is caused by neurons killed by malfunctions in mitochondria -- the submicron-sized power packs found in every animal cell.
These malfunctions are the most immediate cause of afflictions like Parkinson's, Huntington's and Alzheimer's diseases. Malfunctioning mitochondria have also been linked to battlefield aftereffects caused by radiation or by nerve agents like sarin.
But because mitochondria are so small, averaging a few hundred nanometers, scientists have been unable to study them in vitro with the necessary precision to determine the best possible neuroprotectants.
Researchers at the Department of Energy's (DOE) Sandia National Laboratories and the University of New Mexico School of Medicine are using a unique biolaser operating in the nanometer range that has demonstrated the first-ever technique for studying the reactions of such ultrasmall biological organelles in their functioning state. The laser has already shown it can obtain clear signals from individual mitochondria in vitro. The laboratory mitochondria will be rapidly coated by neuroprotectant drugs and then subjected to hostile circumstances.
"'Waterproofing' the mitochondria with specific protectant drugs would increase the survival chances of the brain," says Marcus Keep, a neurosurgeon professor at the University of New Mexico (UNM) School of Medicine.
"Our goal is make the brain less susceptible to diseases like Lou Gehrig's," says Sandia researcher Paul Gourley, a physicist who grew up in a family of doctors.
MITOCHONDRIACS: Surrounded by MRIs of human brains, Sandia researcher Paul Gourley, left, and Marcus Keep, a neurosurgeon professor at the University of New Mexico School of Medicine (UNM), examine a fanciful drawing of a mitochondrion by the late Swedish artist Oscar Reutersvar. The photos are at UNM. (Photo: Randy Montoya)
Preliminary work thus far has shown the biolaser (which recently won first place in the Department of Energy's annual Basic Energy Sciences competition for using light to quantify characteristics of anthrax spores) is able to measure mitochondrial size through unexpected bursts of light given up by each mitochondrion. The laser, using the same means, can also measure the swelling effect caused by the addition of calcium ions -- the reaction thought to be the agent of death for both mitochondria and their host cells. The researchers expect to introduce neuroprotectant drugs into experiments this month, and to be able to test hundreds of possible protective substances daily.
"If we can use this light probe to understand how mitochondria in nerve cells respond to various stimuli, we may be able to understand how all cells make life or death decisions -- a step on the road, perhaps, to longer lives," says Gourley.
To do that, he says, scientists must understand how a cell self-destructs, which means understanding how mitochondria send out signals that kill cells as well as energize them.
The universal energy provider
Mitochondria have long been known as the mechanisms that produce ATP, the universal energy driver for animal life. ATP powers each cell, similar to the way that gas powers each automobile. But scientists have found that the tiny power plants have another function. When cells are signaled to die -- acceptably, as when biomaterial is shed from a uterus during its periodic menstrual cycle, or unacceptably, as the result of certain neurological diseases -- an excess of calcium ions and free radicals that result from certain chemical reactions in the body open a large pore in the inner membrane in that cell's mitochondria. The pore enables release of a protein called cytochrome C that kills the cell. Meanwhile, the mitochondrion itself swells and explodes. One way to stop this suicidal process would be to find a chemical that would shield the mitochondria from these intruders.
Unexpected bursts of light
The observation technique developed at Sandia to test for such effects came about almost by accident. In the innovative lab arrangement already developed by Gourley's group, a micropump sends fluids containing suspect material through a submicron-sized lasing cavity. The cavity is formed between a light-emitting semiconductor and a reflective mirror.
The group expected to push fluid containing mitochondria through the device and to see very weak signals emanating from the tiny organelles. Had this been true, signal-averaging techniques would have been necessary to generate a generalized, necessarily less crisp estimate of responses.
"We were pleasantly surprised but puzzled to see very large signals from each mitochondrion," Gourley said. "A statistical average was unnecessary."
The researchers realized that each mitochondrion acted as a lens for light passing through it because the organelle had a higher index of refraction (1.42) than water (1.33). Light refracted into the mitochondria in effect emerged amplified. It was analogous to a lens concentrating light passing through it.
"When a critical concentration of emitted photons is reached," says Gourley, "stimulated emission of additional photons occurs in the semiconductor."
These photons, as well as those reflected from the mirror, retrace their paths back through the mitochondria. "Wildly wayward photons are lost," Gourley says. "Only the photons that pass back through the tiny mitochondrion will arrive back at the semiconductor with the proper phase and location where the photon amplification (gain) can recur."
This discovery suggested the laser cavity be set up sensitively -- like a gun on a hair-trigger -- by carefully setting the power of an external pump laser that beams energy into the cavity. When a mitochondria cell is present, the light in the cavity reaches critical concentration to trigger the avalanche of photons necessary for laser action.
Thus the tiny organelle becomes the center of a lasing process that yields light signals as bright as that emitted by an entire cell several orders of magnitude larger, offering possibilities for analysis that light scattering -- the current method of choice for rapid mitochondrial analysis -- lacks.
Because the light has to squeeze through such a tiny object, a process Gourley calls "nano-squeezing," the lasing spectra are dramatically altered, which makes cell identification and detection easier.
UNM's Keep, who is also CEO of the Albuquerque-based Swedish-American company Maas BiolAB, has contributed the neuroprotective agent Cyclosporin A, for which his company holds a patent. According to Keep, Cyclosporin A does "waterproof" the mitochondria, but not well enough. The idea here is to use the Sandia biolaser to establish a benchmark for performance against which to measure other, potentially even more effective drugs.
"Cyclosporin protects mitochondria better than anything else known, but it is not a perfect drug," says Keep. "It has side effects, like immunosuppression. Unrelated drugs may have a similar protective effect on mitochondria. Gourley's device will lead to a rapid screening device for hundreds of cyclosporin derivatives or even of chemical compounds never tested before."
While testing with conventional methods would take many people and many batches of mitochondria, says Keep, the nanolaser requires only tiny amounts of mitochondria and drug to test.
"With one tube on the left flowing in a number of mitochondria per second, and microliters of different drugs in different packets flowing in to join them on the right, we could rapidly run through hundreds of different compounds. Each mitochondrion scanned through the analyzer would show if there were a change in its lasing characteristics. That would determine the effectiveness of chemical compounds and identify new and even better neuroprotectants."
Currently, he says, only a few materials can be tested each day. Mitochondria with and without neuroprotectant would have calcium ions added to the mix to see the effect of each potential drug.
The work is funded by DOE's Basic Energy Sciences program, DOE's Office of Biological and Environmental Research and Sandia's Laboratory Directed Research and Development funds.
Keep has also applied for a grant from the US Congress to develop treatments based on Cyclosporin A to help Gulf War victims who develop the neuron disease amyotrophic lateral sclerosis (ALS). ALS, or Lou Gehrig's disease, is a neurodegenerative disorder affecting both Gulf War veterans and civilians that kills motor neurons, causing paralysis and death in three years.
For more information, visit: www.sandia.gov