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Why Do Our Brains Betray Us?
Microarrays: Chipping Away at the Mysteries of Alzheimer's Disease

by Jeff Worley

Genes, like enzymes, are also role-players, and molecular biologists continue to spend long hours in the lab working to shed light on these various roles. Traditional methods in molecular biology generally work on a tedious one-gene-in-one-experiment basis, which does not allow for a whole picture of gene function. However, advances in technology are now paving the way for biologists to study genes on a much larger scale.

Photo of Philip LandfieldUtilizing a new technology called DNA microarray, Philip Landfield (left) and his team, which includes Kuey-Chu Chen and Eric Blalock, in the UK College of Medicine are analyzing thousands of genes and genetic pathways to better understand "incipient" Alzheimer's—the earliest stage of the disease.

"In the past several years a new technology, called DNA microarray, has revolutionized how we do gene research," says Philip Landfield, professor and chairman of the Department of Molecular and Biomedical Pharmacology in the UK College of Medicine. An array, he explains, is an orderly arrangement of gene probes on a small chip. "What this new technology allows us to do is measure 20,000 genes simultaneously and to look at entire genetic pathways," he says.

This work starts with what looks like, but isn't, a simple chip. DNA chips such as the human one used in Landfield's current study, which is focused on better understanding incipient Alzheimer's disease, are fabricated using a process called photolithography (the same technology used in the manufacture of high-performance computer chips like Intel Pentium processors).

In Landfield's project, human brain tissue from UK's ADRC was used to analyze gene activity based on chips made by Affymetrix, a Santa Clara-based company. The UK group analyzed the chips in the university's MicroArray Core Facility, which houses complete gene chip instrumentation. Kuey-Chu Chen, a member of the team involved in this study, directs this facility.

To understand how each of these genes is detected on the chip, we have to dust off some basic terminology from Genetics 101.

Information about protein structure that is encoded into DNA is transcribed into complementary (closely related) RNA structure. These RNA transcripts convey this key information from the nucleus to protein-production sites (ribosomes) in other parts of the cell. This RNA becomes a sort of messenger, and is aptly called messenger RNA (mRNA).

These mRNA molecules bind naturally and specifically to their complementary DNA templates, like a key in a lock. A microarray works by exploiting this union. The probes on the surface of the chip act like a field of thousands of keys, all waiting for the unique locks for which they were specifically made. When a key fits in a lock, binding occurs, and it can be detected using laser scanning technology.

"When a laser is focused on a highly bound region of the chip, it shines more brightly than on an unbound region, so by measuring the light intensity, we can quantify the amount of binding and, therefore, the amount of mRNA in the tissue," Landfield explains. In this way, the researchers can determine which genes are turned on and to what degree.

"By using an array containing many complementary probes to which a subject's tissue is added," Landfield says, "we can determine in a single experiment the expression levels of thousands of genes.

"This is the wonderful thing about microarrays," he adds. "They provide us new ways of dealing with complexity because they allow overviews of the simultaneous activity of multiple cellular pathways."

Recently, Landfield's group has used this technology to better understand incipient Alzheimer's. "'Incipient' means the very beginning of the disease regardless of age," Landfield explains. "This earlier time point is extremely vital to study."

And this is where microarrays come in. This technology recently allowed Landfield and his team to analyze the simultaneous activity of multiple cellular pathways in the hippocampus of 31 brain samples—22 Alzheimer's subjects of varying severity and nine "controls." All of these brain samples were obtained through the ADRC's BRAiNS program.

An article on this work by lead author Eric Blalock (pharmacology) appeared last month in Proceedings of the National Academy of Science. In what co-author Landfield calls "a welcome comment" on this article, one reviewer said, "the UK group has convincingly shown us a new model, in what is the largest and best microarray study of Alzheimer's disease; the idea of correlating gene expression with pathology is unprecedented in this kind of study." Article co-authors included Landfield, Jim Geddes (Spinal Cord and Brain Injury Research Center), Chen (pharmacology), Nada Porter (pharmacology), and Markesbery.

Two basic indicators of Alzheimer's disease were initially examined in this study: neurofibrillary tangles (NFTs) in the hippocampus and scores on the standard MiniMental Status Examination (MMSE). All of the brain samples had previously been characterized by Markesbery, Geddes and other collaborators, who routinely do brain autopsies at Sanders-Brown, to determine brain pathology. NFT density was given a "score" in order to quantify the amount and type of brain deterioration.

Second, each subject's record on the MMSE was examined and used as a cognitive marker of decline. This test represents a brief, standardized method by which to "grade" cognitive mental status. It assesses orientation, attention, immediate and short-term recall, language, and the ability to follow simple verbal and written commands. It also provides a total score that places the individual on a scale of cognitive function. The researchers therefore had two ways to quantify the severity of Alzheimer's-related dysfunction in each subject: mental decline as measured with the MMSE and pathological changes in the brain as measured by NFT.

"The reason we took this strategy instead of initially putting the known Alzheimer's subjects into groups (incipient, moderate or severe) versus the control group is—and I can't emphasize this enough—it's hard to tell when someone has incipient Alzheimer's," Landfield says. "It's very easily confused with normal aging."

So the researchers ignored whatever diagnosis the subject might have had and, instead, used a statistical correlation strategy to define what "incipient" meant. "We correlated each gene with how many NFTs there were, and we did the same for the cognitive scores," says Blalock, who did the number crunching in the office next to Landfield's. "We subsequently identified thousands of genes that correlated with one or the other marker in all 31 subjects."

"But the most important thing to come out of this is that we also identified hundreds of genes that correlated with the early phase of Alzheimer's," Landfield says. "We were then able to assign identified genes to biological process categories to get an idea of which cellular pathways were being turned on." One important discovery the group made was that incipient Alzheimer's is what Landfield calls "a genomically orchestrated phenomenon."

"Researchers knew some genes were turned on or off, but this is the first study to find hundreds of these genes that correlated with incipient Alzheimer's."

"We've now defined pathways that are activated or repressed, and this knowledge can become a tool to diagnose—and predict—who's going to develop incipient Alzheimer's disease," Blalock says. Landfield emphasizes the fact that in order to make such a diagnosis, scientists wouldn't need much tissue. "Brain biopsy is a possibility," he says. "It's done in some brain cancer patients."

"Another possibility," Blalock says, "is a blood test. White blood cells and specialized brain cells called microglia have a lot in common. Certainly some of the signals that we are measuring in our study could have originated from microglia. If these signaling pathways are activated in parallel in the brain and blood of Alzheimer's patients, then a diagnosis could be made using microarray-based blood tests."

"Of particular importance, these genes and genetic pathways can now become targets for new drugs," Landfield adds. "We hope that, by using these kinds of genetic markers and patterns, it'll become much easier for pharmaceutical researchers to develop these drugs.

"The ultimate goal," says Landfield, "is to intervene earlier so that we can slow the progression of this terrible disease, or possibly stop it in its tracks."

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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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