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Why Do Our Brains Betray Us?
How Do Enzymes Fit into the Alzheimer's Puzzle?

by Jeff Worley

While Scheff is busy studying synaptic connectivity, Louis Hersh is working to better understand another kind of connection: enzyme activity and amyloid beta (A-beta) protein deposits in the brain. In dozens of projects both outside and inside of a living organism, scientists in the past 20 years have found that A-beta protein deposits are a major component of senile plaques in Alzheimer's-diseased brains. These plaques and neurofibrillary tangles (shriveled strands resembling bundles of straw inside damaged neurons) are strong signatures of the disease. Recent data suggests that the A-beta protein may also be toxic to cells before it forms deposits.

So how do you stop A-beta deposits from forming?

Photo of Louis HershLouis Hersh in UK's College of Medicine is focusing his work on two enzymes—neprilysin and insulysin—that team up to clear away dangerous amyloid beta deposits in the brain.

Hersh, professor and chairman of the Department of Biochemistry in UK's College of Medicine, believes that two enzymes—neprilysin and insulysin—might help do exactly that. "These two enzymes have received the most attention as A-beta-clearing proteins," he says.

Like firefighters off at the alarm bell, enzymes marshal their forces when the call comes. Their job is to move to the scene of action as quickly as possible to start chemical reactions the body needs to stay in balance and keep going. Enzymes are enablers.

The most recent and compelling evidence that neprilysin is an A-beta regulator comes from research with "knockout" mice, who have had this enzyme removed. "In these mice, A-beta peptide levels go up," says Hersh, who has studied neprilysin for over 25 years through a variety of projects. "We take this as evidence that neprilysin normally breaks down these A-beta peptides, and when the enzyme isn't there or decreases in amount, the A-beta levels go up, and that's not good." He adds that although mice don't naturally make amyloid deposits as the human brain does (which is probably why mice don't get Alzheimer's), many of the same enzymes, such as neprilysin, are present in both mice and humans.

Insulysin has also shown itself in the past few years to be a mighty A-beta regulator, so Hersh's lab has turned its attention to this first cousin of neprilysin (both have a metal ion and contain some zinc).

"Enzymes work on and break down small molecules, and whatever an enzyme goes to work on is called its substrate," Hersh explains. "For example, digestive enzymes work on many substrates—think about the variety of food you eat in one day." The A-beta peptide, it turns out, is a substrate for insulysin, so insulysin and neprilysin have a common enemy.

One very important recent finding in Hersh's lab is that these two enzymes work to stop A-beta in different but complementary ways.

"The reason neprilysin has been so widely studied is because it's one of the few enzymes that work on the surface of the cell, on the cell membrane. A-beta peptides are secreted from the cell. So neprilysin can go to work on A-beta as it's being secreted or as it's beginning to travel into extra-cellular space." Insulysin, on the other hand, works inside the cell, so there's a sort of tag-team approach to stopping A-beta in its tracks.

It used to be thought, Hersh says, that as we age we overproduce A-beta, that it overwhelms the cell. There is evidence now to the contrary.

"We're starting to believe that rather than an overproduction, what's happening is an 'under-clearance,'" says Hersh. "The activity of the enforcer enzymes is somehow lessened, which allows the damaging effects of A-beta to go relatively unchecked."

This realization has moved Hersh's work into a new direction, he says. With continued funding through the Alzheimer's Association and the National Institute of Aging, he's looking now at various ways to increase the activity in the brain of these two key enzymes.

"We've never lost sight of the potential clinical applications of what we're doing," Hersh says. "We're looking at a number of approaches to utilize these enzymes to break down A-beta both in the brain and elsewhere in the body. We're just starting work with a biotech company to screen chemical libraries to see if we can find drugs that will activate insulysin. That's what I'm most excited about right now." Hersh hopes this research will lead to clinical trials in about five years.

Jeffrey Keller never thought his years of research training would lead to an intense study of garbage, but, he says, that's just what's happened.

"Cells, like people, generate garbage, and all cells have to remove it. In order to keep this clutter at a non-dangerous level, cells rely on one particular intracellular enzyme, called the proteasome," explains Keller, a UK assistant professor of anatomy and neurobiology whose research at Sanders-Brown is focused on understanding the work of this essential enzyme. Think of proteasome as the cell's chief garbage collector.

The garbage under scrutiny is mainly oxidized or mis-folded proteins, and since accumulations of this substance have been implicated in Alzheimer's disease, Huntington's disease and stroke, Keller's research might help provide important clues to the etiology of these diseases.

Scientists think they have a handle on what the enzyme generally does but very little is known about the exact expression of proteasome activity in the normal and diseased brain, and even less is known about the possible role of proteasome inhibition in cell death, says Keller, who spent one year as a postdoc at Sanders-Brown after earning his doctorate here in molecular and cellular biology in 1998.

"Our lab is trying to understand exactly what the garbage disposal in the brain looks like when it's operating normally and what it looks like when it goes bad. In addition, we're interested in understanding what causes it to stop functioning normally as we age, or in age-related disease."

In his studies Keller uses basic molecular techniques, called the reverse transcriptase polymerase chain reaction (or RT PCR), in cellular, animal and human tissue.

"Using RT PCR, we are able to visualize or quantify the expression of individual proteasome subunits," Keller says. "This is a common way of investigating gene expression. When we couple this with our ability to isolate and purify the proteasome enzyme, we have very powerful techniques for exploring the proteasome in aging and age-related disease."

In this work, he and his co-investigators are utilizing yeast, which, he says, looks a lot like human brain tissue. "Remarkably, the pattern of proteasome expression and the process of oxidative stress are similar in yeast and human tissue. The advantage of using yeast lies in the power of genetics—we can rapidly and directly test the role of specific genes in regulating the proteasome and its toxicity in a matter of days instead of months. We can then apply this knowledge to the animal models of disease, and even the actual tissue from human disease."

Keller admits that this approach is extremely challenging, not because of their methodology but because of what he calls "the numbers game."

"Proteasome is so complex. At its heart, it's a structure made up of 28 different proteins. There are at least 82 additional proteins that can combine in different ways to comprise this 28-protein complex. Every cell has a smorgasbord of choices to assemble its garbage disposal." The bottom-line goal of this work, he says, is to differentiate the changes that occur with normal aging from the changes that occur with Alzheimer's disease.

Keller says his lab is making good progress in "getting proteasome to give up its secrets," and that any success is due to the various collaborations in the Center on Aging: Allan Butterfield (chemistry and Center of Membrane Sciences), Harry Levine (Center on Aging), Rodney Guttman (gerontology Ph.D. program), and Jim Geddes (anatomy and neurobiology). "They're experts in different areas of neuroscience, and we are able to apply their skills to our project. And of course Dr. Markesbery, who's at the center of all of the work we do here," Keller adds.

Keller is at the halfway point in this work, which is being supported by the National Institute of Aging for $1 million for four years and by other grants from the National Institutes of Health and private foundations.

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For more information about Alzheimer's disease clinical trials at the UK Sanders-Brown Center on Aging, call 859/323-6729 or visit www.mc.uky.edu/coa.

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