Alzheimer's: Fighting Back
UK researchers are working at the cell level to discover the pathways of the disease and how to counter it
About a quarter of a century ago, a new word entered public usage: Alzheimer's. Named after the German physician Alois Alzheimer in 1907 and long-used in medical circles, the word put a label on the dehumanizing condition of degenerative memory loss and physical deterioration which ultimately leads to death. An estimated 14 million Americans will have Alzheimer's by the year 2020.
At the University of Kentucky, researchers began working to understand this disease in the mid 1970s, according to William Markesbery, director of the Sanders-Brown Center on Aging. "I worked with Bill Ehmann in the chemistry department here for over 20 years, and we had our first grant to study Alzheimer's in 1976 or '77," Markesbery recalls. "This was the first grant at UK, and at this time there weren't many grants around the country to study the disease."
William Markesbery, one of the first scientists at the University of Kentucky to do research into Alzheimer's disease, performs autopsies on brains donated to UK's Alzheimer's Disease Research Center. "Only through an autopsy of the brain can it definitely by determined whether someone had Alzheimer's or not," Markesbery says.
In the 1960s and 1970s the study of Alzheimer's disease focused on describing the effects of the disease, trying to describe what Alzheimer's "looked like." "Back then it was very descriptive," Markesbery says, "but all the descriptive work is behind us and now we're at the molecular level trying to understand the mechanisms of the disease. And," he adds, "it's gotten to be very exciting."
Markesbery is now a member of a research team, which includes Chemistry Professor Allan Butterfield and Mark Mattson, a neurobiologist and professor at UK's Sanders-Brown Center on Aging. Four years ago Butterfield, Mattson and John Carney (then a professor of pharmacology at UK) made an exciting discovery that captured international attention: they discovered how brain cells are killed in the process of Alzheimer's disease, a finding that National Institute on Aging director T. Franklin Williams called a "breakthrough in the understanding of the disease."
Butterfield was instrumental in developing at that time what he called the "molecular shrapnel model" of brain cell death in Alzheimer's disease.
"I began thinking about the beta-amyloid protein, a peptide [a series of amino acids] that is the core block of abnormalities in the brain called plaques," says Butterfield, who also directs UK's Center of Membrane Sciences. "Plaques are one of three features always found in the brains of Alzheimer's victims. The odd thing about this peptide in plaques is that no matter how you try to dissolve it, you can't do it. As a scientist, I asked myself, 'How can you account for this?' The only answer I could come up with is that the peptide was forming covalent bonds; very strong chemical associations. 'And how are covalent bonds formed?' I asked. The answer that occurred to me is by peptide free-radical reactions."
Free radicals, Butterfield explains, are molecules with one or more unpaired electrons, an imbalance that causes them to be extremely unstable and reactive. "Nature abhors having unpaired electrons," says Butterfield. "So free radicals are very reactive because they are always trying to either give up or accept an electron." Butterfield, Mattson and Carney set to work immediately to see if they could detect free-radical activity from the beta peptide. After several control experiments using electron paramagnetic resonance spectroscopy, the researchers found that this villainous peptide was, in fact, associated with free radicals. The free radicals attacked the cell membranes, substantially damaging them.
During the last 10 years, Mark Mattson has become a leader in studies of the mechanisms of neuronal deregulation.
Recent Findings about Cell Membrane Death
That was four years ago. Armed with the first theory that put Alzheimer's disease into an understandable and verifiable framework, Butterfield, Mattson and Markesbery (Carney left UK for a job in private industry) subsequently found that the beta peptide causes nerve cell damage by inducing a free-radical-mediated process called "lipid peroxidation" in the nerve cell membrane. Lipids are fatty or waxy substances which constitute, along with proteins and carbohydrates, one of the principal components of living cells.
"We found that such lipid peroxidation results in the impairment of protein function in the membrane that normally transports ions (sodium and calcium) out of the cell and glucose into the cell," says Mattson. "This results in excessive elevation of intracellular calcium levels and reduced levels and reduced energy production in the neurons, rendering them vulnerable to being 'excited to death.'"
Mattson says this finding explains the group's previous discoveries showing that the beta peptide disrupts calcium regulation in neurons, making them sensitive to degeneration.
More recently, the researchers have focused on one of the products of the free-radical attack, a molecule called 4-hydroxynomenal, or HNE. This molecule was found to be elevated in Alzheimer's brains. The scientists believed that this extremely reactive molecule, which targets amino acids (the chief components of proteins) on the cell membrane, caused their dysfunction. HNE, they thought, led to the membrane being weakened, a condition which subsequently allows calcium to leak into the cell and kill it.
Mattson showed in 1996 that if amyloid is introduced into neuronal cells in culture that HNE will be formed, and Butterfield showed in 1997 that if HNE is added to brain cells, the conformation of cell membrane proteins will be changed.
"This finding confirmed an earlier discovery of ours-- amyloid's effect on various transport proteins," says Butterfield. "This was a very, very good finding," he adds.
"This result, that amyloid was associated with free radicals, also might explain another enigmatic feature of Alzheimer's disease," Butterfield says. "We see many reports in the scientific literature of different proteins and lipids being altered in this disease." Butterfield proposed that amyloid-associated free radicals, being reactive, would bind to these transport proteins and alter their function in Alzheimer's brain tissue.
"These free radicals also weaken the structure of membrane lipids," he says. "Together, these free-radical-induced effects on membranes might explain the altered enzymes, transport proteins and lipids in the brain."
Most recently, the researchers have focused their attention on a molecule called glutathione, a tri-peptide normally found in everybody's cells. An important characteristic of this molecule is that it naturally counteracts HNE, preventing it from damaging transmembrane proteins.
Using high-performance liquid chromatography, Allan Butterfield separates peptides found in the Alzheimer's disease brain.
"We have just completed a study in which we chemically reduced the glutathione level in a living animal--we used gerbils--with the question in mind: With a lowered glutathione level, would the oxidated stress be worse than it would be with the regular level of glutathione present?" Butterfield says. "And the answer is yes. And then we turned the question around: If we increase the glutathione level, would there be increased protection? And the answer is, again, yes."
This is an important discovery, Butterfield says, because it means that the body's natural antioxidant system might be used to fend off free-radical damage. If the level of this molecule could be significantly increased in our bodies, we would have a natural system of damage control.
Calcium's Role in Cell Death
In recent years calcium's role in cell death has been hypothesized by researchers. In the early 1980s speculation began to grow that aging alters the calcium balance and this in turn makes the neurons more vulnerable for Alzheimer's disease.
"It's well known that calcium is critical to a wide range of cellular processes," says Philip Landfield, UK professor of pharmacology. "But if calcium becomes, for some reason, deregulated and the level rises, the nerve cell will be gradually damaged because of calcium's toxicity at higher levels." Landfield explains that calcium, a basic mineral we get from some foods and milk, is usually maintained at a very low level by a series of buffering mechanisms and is normally cleared rapidly from the cell.
Backed by a recent $1.4 million NIH MERIT award and a $5 million program project award from the National Institute on Aging, Landfield and a team of scientists at UK's Chandler Medical Center are investigating calcium regulation in aged cells. The effort includes multiple projects, based primarily in pharmacology and the Sanders-Brown Center on Aging.
"We received this MERIT award in part because of our discovery here at UK that there are more calcium channels in the membranes of aged neurons," Landfield says. "We had the essential advanced technology here and the experienced researchers who'd been trained how to use it."
The technology Landfield refers to begins with a powerful microscope that allows the viewer to observe the dissecting of a cell. Using this microscope, Research Associate Eric Blalock can view one nerve cell from an aged rat and then--through a very precise and delicate procedure--study the calcium channels within that cell. Landfield explains: "Every membrane has channels in it. Since we want to study only calcium channels, we introduce drugs known to react with and block other types of channels. Then we attach a small glass pipette with a hole in the tip to the surface of the nerve cell. Now, what's under the mouth of the pipette is all that's recorded, the activity of a single channel or single molecule." A series of blips--peaks and valleys--on a printout records this channel opening and closing.
When told that this all sounds extremely complex and futuristic, Landfield says that this observation is understandable and is half right. It is difficult science, but the method for exposing the cells has been around "for all of seven years or so now," he says, adding that the scientists who developed this method won the Nobel Prize for it in the early '90s.
After the neuron's channel activity is recorded, the cell is pulled up by the pipette and placed in a small test tube. It is then taken to the lab next door, to Kuey-Chu Chen, a research assistant professor. "We want to analyze molecular contents of the cell, but there's a problem," Landfield says. "There's simply not enough material to work with in one cell."
Philip Landfield's research examines the role of calcium in aging brains.
That's where Chen comes in. Her job is to take the DNA from the cell and through a common technique called PCR (polymerase chain reaction) keep recycling (duplicating) the DNA until there's enough to actually work with. This process amplifies the genes in the cell just as turning up the volume on a radio amplifies a song. The result is what looks like, to the untrained eye, a series of darker and darker smudges displayed on a computer screen. These marks tell the researchers how much DNA is present to make proteins the researchers want to study.
"This work focuses on one of the central questions our group is trying to answer: Why are there more of these calcium channels in aging? There are two possibilities," Landfield says. "Either more channels are being made or fewer are being broken down. Either way, it's a deregulation problem that results in a buildup of calcium." Further study of the amplified genes will lead to understanding the activity of these calcium channels, Landfield says.
"Although PCR is an established technique, very few labs in the world do this from a single cell," says Landfield. "But this single-cell amplification allows us to compare young cells with old, to correlate the amount of different gene expression with channel functions and determine the cells' vulnerability to being killed." The ultimate goal is to determine which gene pathways are involved in killing neurons. "What's unique about this work is that we're using new techniques that are only beginning to be available in basic biology and applying them to the problems of brain aging."
Down the hall from Chen's lab, the calcium question is being explored in another way. Olivier Thibault, an assistant research professor, sits in a small semi-darkened room surrounded by an impressive array of equipment. A large microscope with numerous attachments is complemented by two computers that capture different types of data on calcium. A TV screen records a procession of peaks and valleys from, Thibault explains, "the currents from the nerve cell channels under scrutiny."
The pulses that translate on the screen are being recorded from a pipette lodged in the center of the rat brain slice, which, according to Landfield, is a spot that will least affect the normal firing of the cell. "Each time the cell fires, there's an electrical change in the membrane, and it opens the calcium channel," Landfield explains.
Another screen shows the magnified neuron, some of it glowing red. The red dye is a reagent added to the cell. When it comes into contact with calcium, it glows red so that the researchers can see where the calcium is in the cell, and how much there is.
"This confocal laser scanning microscope can show us whether there is calcium present in the cell and, beyond that, we can see where the calcium is in different layers and levels within the cell," Thibault says. "It's very sensitive and difficult to use but is an indispensable instrument for this kind of research." An advantage to this approach, once it is mastered, Landfield says, is that the electrical activity of young cells can be compared with that of older cells, and the amount and location of calcium can be compared. "It's a very powerful method for us to see calcium influx at different depths in the cell," he says.
A few doors down the hall from where Thibault does his work, the calcium question is being explored on another front. Nada Porter, an assistant professor of pharmacology, and Veronique Thibault, Olivier's wife, are working with rat brain cells in culture. This is a type of fast-forward research, Porter explains, because embryonic cells develop the same changes in calcium channels in four to five weeks in a Petri dish as the animal itself develops over a period of years.
"One clear advantage to this," Porter says, "is that since some of the same things are happening in cell culture as happen in the living animal, we may be able to reduce the number of animals we are studying and focus more on the calcium changes in culture that are most responsible for making neurons vulnerable."
"Our strategy has been, and continues to be, to bring the most advanced techniques in the field to the study of aging," Landfield says. "We're using cutting-edge methodology comparable with any in the world to track down the key changes that come with age to make a cell more vulnerable to Alzheimer's disease. When I started on this research 20 years ago, I figured it'd take about 10 years to solve the role of calcium in aging and cell death. I didn't realize how long it would take just to test each aspect of this. I guess 10 years always looks a lot longer when it's in front of you rather than behind you."
This sequence of photos shows a single brain cell being recorded and then collected for molecular analysis. In A) a patch pipette is used to record the electrical activity of the nerve cell's calcium channels. B) The neuron is gently pulled away from the main tissue slice. C) The neuron is transferred from the patch pipette to the harvest pipette. The harvest pipette is removed from the bath and its contents placed into a collection tube for analysis of the cell's profile of gene expression. The researchers hope to identify how genes regulate activity of the cells and eventually determine how this gene expression changes with aging.
Alzheimer's Disease Research Today
In contrast to the research climate for Alzheimer's 20 years ago, there are now hundreds of projects worldwide that are focusing on the disease and how to understand it and control it. One reason for this mushrooming of projects is that the largest population group in the country, the Baby Boomers, are now dealing firsthand with the disease as they see mothers and fathers, aunts and uncles, suffer and die from Alzheimer's. Other reasons include population growth and longevity.
"We just have so many more people now," says Markesbery, "and the elderly generation is fairly outspoken and has a major political impact on our congressional leaders. You know, there's nothing more frightening for an older person than the thought of losing his ability to remember and to think and reason clearly; so there's a large and vocal group of folks in the over-65 bracket lobbying very strongly to do something about this disease."
Alzheimer's researchers are pleased about such groups as the AARP (American Association of Retired Persons), because the pressure they bring to bear on political leaders translates directly into more federal funds for research into Alzheimer's. "The money we get from agencies like NIH also helps support graduate students and post-docs to be trained how to do good science," says Butterfield, "and Kentuckians should be proud that we're training first-class students how to do first-class science."
The short-term goal for Butterfield and his research team is to continue to try to understand the HNE molecule, to pinpoint its role in damaging healthy brain cells. Butterfield says they will better understand how these cells die based on the free-radical model and how to prevent that damage mostly through further exploration of the glutathione molecule and other brain-accessible antioxidants.
"As a consequence, we're going to gain a lot more insight into how to limit or prevent the damage associated with Alzheimer's disease, our overall goal," Butterfield says.
Vitamin E's Protective Role
There have already been payoffs from Alzheimer's research, including recent studies that show the effectiveness of the much-heralded vitamin E to gobble up free radicals. "We've published two papers that show vitamin E is protective against free-radical damage," says Butterfield. "And another study, by a group of 12 clinical scientists who published their work recently in the New England Journal of Medicine, showed that high-dose vitamin E given to late-stage Alzheimer's patients prevented their institutionalization up to seven months. Such studies are important in themselves and also important to our work since this is further proof that our concept about free-radical oxidative stress may be accurate."
High-dose vitamin E, he explains, means 2,000 units a day, which Butterfield says is beyond what someone not afflicted with Alzheimer's should take. "Though I have to toss out the caveat here to see your doctor before deciding how much vitamin E to take, I take a thousand units a day myself."
Markesbery, who besides being a researcher into Alzheimer's is a well-respected clinician, says that the strategy in the '90s is to treat the disease before it starts in those at risk, a sort of neuroprotective therapy.
"Vitamin E is clearly beneficial if taken in the right dosages," he says. "And there are other compounds such as non-steroidal, anti-inflammatory drugs. Estrogen seems to slow free radicals down. And of course we're working now on the possibility that part of the body's own machinery--glutathione--can fight off free-radical damage. And I'm certain there are other things that are going to come along, too."
"With this recent news about vitamin E and various other therapies, I think it's reasonable to believe that we will soon be delaying some of the more deleterious effects of Alzheimer's," Butterfield says. "And if you could delay them for even a year or so, think of the enormous benefit in terms of lifestyle and daily living for these patients and their families."
Mattson, in a recently published article which appeared in Science & Medicine specifies what these "other therapies" are. "In addition to taking vitamin E and vitamin C, measures can be taken to forestall Alzheimer's disease," he says. "You should restrict your dietary intake to 1,600 to 2,000 calories per day throughout your adult life. Why? Because a number of studies have shown that reducing calorie intake also reduces cellular oxidative stress."
It's also important, Mattson says, to sustain a high level of intellectual activity and vigorous physical activity. And postmenopausal women, he adds, should consider estrogen replacement therapy.
"These recommendations probably sound familiar, because they are also the approaches that reduce the risk of developing cardiovascular disease and cancer," Mattson says. "The reason is straightforward. Cellular oxidative stress plays a major role in all of these age-related disorders. Reducing the level of oxidative stress reduces the risk of each disorder."