Three scientists leverage brain trust, collaboration in combating Alzheimer's

Competition for grant money and publications has traditionally led to a guarded professional relationship among researchers. Lately, however, a new spirit of cooperation has begun to take hold. Although the former approach had a measure of success, and still persists in some circles, researchers are beginning to realize that some problems are too complex to be solved single-handed. They call for tools and perspectives from multiple disciplines, and for the free flow of information.

It was in this spirit that Drs. Dietrich Stephan, Eric Reiman, and Joseph Rogers proposed an ambitious, large-scale collaborative effort to investigate cellular changes associated with Alzheimer's disease; Their proposal, a collaboration among researchers at the Translational Genomics Research Institute, Sun Health Research Institute, Banner Good Samaritan Medical Center, and Mayo Clinic Scottsdale, was awarded $2.5 million in grant funding by the National Institute on Aging.

Alzheimer's is primarily a disease of the elderly, one that results in memory and thinking problems due to progressive loss of cells (neurons) in the brain. Symptoms can include impairments in memory and learning, language skills, judgment, and the ability to perform routine tasks. Associated problems can include disorientation, personality change, agitation, and other behavioral problems. The disease is progressive and irreversible, and no cure is currently known

Such a simple definition, while essentially accurate, belies the disease's underlying complexity, and the complications that arise when trying to study it. Fortunately, three of Arizona's most respected research scientists are on the job.

TGen's Dietrich Stephan will contribute his expertise in utilizing microarray technology to study genetic disorders. Joseph Rogers, whose Sun Health Research Institute leads Arizona in Alzheimer's grant money and publications, will bring his extensive experience in studying the contributing role of brain inflammation in Alzheimer's. SHRI will also furnish the study with access to its remarkable brain donation and preservation facilities. Eric Reiman of Banner Good Samaritan will share his breakthrough research in measuring brain chemistry changes via Positron Emission Tomography, a technique that has proven useful in studying young people who possess an Alzheimer's susceptibility gene.

They will need every weapon in their arsenal if they are to understand the genetic mechanisms of this complex disease.

In many other disorders, there is a clear relationship between a genetic defect and the symptoms of the disease. In the simplest case, a single gene provides the instructions for making a particular protein. When there is a change in the gene sequence "instructions," there is also a change in the corresponding protein. Each protein has a specific function that it performs in the cell; a change in the protein may cause it to fail in that function, leading directly to disease symptoms.

Alzheimer's disease appears to be much more complex than this. Instead of a single causative gene defect—which does indeed account for a relatively small number of cases which affect family members before the age of 60—the disorder may involve several factors, such as the combination of normal gene variants in a patient, effects of the normal aging process, and various environmental influences.

While the role of each of these elements is difficult to tease out, researchers do have some clues to use as starting points. Among these are the microscopic protein deposits called "tangles" and "plaques" that are found in the brains of Alzheimer's patients. Just what causes these deposits, and what effect they have on neuron degeneration itself, are two of the key questions in Alzheimer's research today.

"Plaques and tangles have been a part of the research since Alois Alzheimer performed his famous autopsy in 1906, and their presence is still required for a definitive diagnosis of Alzheimer's," said Rogers. "For almost a century, researchers have been trying to figure out what they are and how to get rid of them."

Another clue involves the apolipoprotein E (APOE) gene. There are three variations (called alleles) of this gene found in the population: APOE 2, APOE 3, and APOE 4. A person has two copies of the APOE gene, which means they can have either two copies of APOE 3, two copies of APOE 4, one copy each of APOE 3 and APOE 4, etc.

The APOE 4 allele is a susceptibility gene found in almost one-fourth of the population. In comparison to the other APOE alleles, each additional copy of the 4 is associated with a higher risk of Alzheimer's. However, since this gene doesn't determine precisely whether or when someone might develop Alzheimer's, and it doesn't tell us yet what we might do to prevent the problem, APOE genetic testing is not recommended as a means of predicting a healthy person's risk for developing this disorder.

Not all people with one copy of APOE 4 (up to 23 percent of the population) get the disease, indicating that there may be variants of other genes that, in combination with APOE 4, increase the risk of getting Alzheimer's.

One way to link these clues to their role in the progress of Alzheimer's is to look at the associated changes in gene expression—the extent to which a gene is turned on or off. For example, a protein's function can be altered by a defect in the gene's "instructions" for making that protein; the protein's function is changed because the protein itself is changed. A protein's function in a cell can also be altered by the amount that is made, with an increase in the "expression" of the protein's gene leading to an increase in the corresponding protein.

The change in expression level of one gene can lead to or result from changes in the level of expression of other genes. The pattern of these changes can give researchers a better understanding of the root cause of disease symptoms. For example, an increase in the amount of a certain protein may cause it to deposit into tangles. However, this increase in gene expression may have been in response to the increase/decrease of the expression of a second gene. The underlying cause of neuron loss, as well as the tangles, may then be due to change in the second gene rather than the first.

The collaboration of Stephan, Rogers, Reiman, and their colleagues will focus on studying the gene expression changes associated with Alzheimer's markers, such as plaques and tangles. They will also delve into the association between the APOE 4 allele and chemical changes in certain areas of the brain.

 

The Players

Each researcher brings a unique background and perspective to the project. Stephan brings expertise in gene expression "profiling"; Rogers' contributes his expertise in tissue preservation and well-recognized history of basic research on Alzheimer's disease; and Reiman's offers his clinical experience with Alzheimer's and expertise in brain-imaging technology.

Dietrich Stephan

The significance of the undertaking is not lost on Dietrich Stephan of the Translational Genomics Research Institute. Stephan considers the NIH-funded collaboration to be a "test of how TGen is going to operate in the community." If the institute is to achieve its goal of rapidly translating genomic discoveries "from bench to bedside," he notes, "TGen cannot operate in isolation."

The NIH grant is awarded to TGen, after which half of the money will stay at TGen, while the rest is distributed to research institutes that are already an established part of the Phoenix community.

Now senior investigator and director of TGen's Neurogenomics Division, Stephan has only recently brought his expertise in gene expression analysis to Arizona. After obtaining his Ph.D. in human molecular genetics from the University of Pittsburgh, he worked with Dr. Jeffrey Trent as a postdoctoral fellow in cancer genetics at the NIH's National Human Genome Research Institute, where he later became a senior staff fellow. He is currently an associate professor of neurology at the University of Arizona and an adjunct professor of molecular and cellular biology at Arizona State University.

Dr. Stephan is also chairman of an NIH-funded consortium of genome institutions, centered in Phoenix, that apply state-of-the-art tools to thousands of neuroscience investigators nationwide. The consortium includes that includes Duke University and University of California, Los Angeles.

When microarray technology came on line, Stephan quickly realized its importance as a tool for investigating the mechanisms of genetic disorders and became an expert in the technology. He has applied this expertise to analyzing a variety of genetic disorders, including epilepsy. While serving as co-director of the Microarray Center at Children's National Medical Center, Stephan used array technology as a diagnostic tool for various types of leukemias.

Microarray technology involves fixing known gene sequences onto a chip, making a "gene array." Researchers prepare cells with a desired or interesting gene expression profile to allow the mRNA (copies of the gene instructions described above) to bind to the gene sequences on the chip. The mRNA copy of a gene will bind to the specific gene sequence that it was made from, generating a color signal; a brighter signal means that more mRNA is bound, indicating that there is more of that particular mRNA in the sample. Current microarray technology allows the determination of gene expression levels of 20,000 genes at one time. %pagebreak%

For the proposed studies, Stephan's group has had to master another cutting-edge technique: laser capture microdissection, which allows researchers to selectively pick out a single type of cell from a tissue sample. For example, scientists working with a complex mixture of cells could opt to remove only those neurons containing tangles. By comparing the gene profile of these cells with that of the same type of cell from a person without Alzheimer's, researchers will be able to determine which genes are increased or decreased in expression (have more mRNA in the sample) when tangles are present.

These fairly new techniques, along with advances in the ability to diagnose Alzheimer's, make the NIH-proposed experiments possible.

Stephan views Arizona as a "mecca for neuroscience research." Along with the clinical and basic neuroscience strengths that comprise the state-supported Arizona Alzheimer's Research Center (AARC) and the NIH-sponsored Arizona Alzheimer's Disease Core Center (ADCC), there are opportunities for collaborations on other neurological disorders, along with a multitude of researchers at Barrow Neurological Institute, ASU, UA, and Banner Health.

Currently, Stephan is working with Tim Miller at the University of Arizona on amyotrophic lateral sclerosis (also known as Lou Gehrig's disease), and with the Sun Health Research Institute and the Mayo Clinic Scottsdale on Parkinson's disease.

Eric Reiman

Eric Reiman is credited with initially bringing the three players to the table to discuss a possible collaboration. As director of the AARC and ADCC, he is very familiar with the role of a facilitator. Indeed, this study capitalizes on additional collaborations with the NIH-sponsored Alzheimer's Disease Centers at Washington University in St. Louis and Duke University, which are contributing additional donated brain samples to the Arizona research team, and the National Alzheimer's Coordinating Center at the University of Washington, which is helping to coordinate activities among the three Alzheimer's Disease Centers.

The AARC and ADCC involves a consortium of eight Arizona institutions dedicated to the understanding, treatment, and prevention of Alzheimer's disease (the consortium is frequently referred to as the Arizona Alzheimer's Disease Consortium, but this name has not been officially approved). According to Reiman, the consortium was formed from a combination of "good will and scientific desperation to capitalize on complementary scientific resources in the state."

Input from multiple disciplines and organizations is essential for dealing with a problem of such scope and complexity, and the ADC has become a nationally recognized model for "leveraging complementary resources" in its approach to biomedical research. The goal of the Arizona consortium is to create a "statewide Alzheimer's laboratory without walls."

Reiman is a tenured professor and associate head of psychiatry at the University of Arizona. He is also clinical director for neurogenomics at TGen and sits on the board of directors of the Flinn Foundation.

Of his many roles, it is that of scientific director of the Positron Emission Tomography (PET) Center at Banner Good Samaritan Medical Center that has gained him recent attention. Reiman and colleagues reported changes in the brain chemistry of healthy people in their 20s and 30s that had one copy of the Alzheimer's susceptibility APOE 4 allele—more than four decades before the possible onset of memory and thinking problems. This finding promises, among other things, to provide new information about the earliest brain changes involved in the development of Alzheimer's, as well as new opportunities for its prevention.

Reiman's team detected these changes using PET imaging. In this technique, a "tracer" molecule containing a radioisotope is given to a patient, and the low level of radiation emission is detected and used to create an image of the patient's brain. Reiman's imaging studies revealed that in certain areas of the brain the breakdown of glucose was slower than that seen in scans of people without the APOE 4 allele. The NIH study will involve gene expression profiling of the areas of the brain that showed this reduction.

Reiman has worked on PET imaging since 1983, when he came to Washington University at St. Louis, the birthplace of the technique. His more recent work has been aided by the efforts of his collaborators at Banner Good Samaritan and Arizona State University, who have contributed to the development of powerful software for the molding, averaging, three-dimensional visualization, and statistical analysis of PET and MRI brain images and their application to the study of Alzheimer's disease.

TGen, too, receives praise from Reiman, who has characterized the institute's technical expertise and "generosity of spirit" as a boon to Arizona. He is excited about the possibilities that have opened up in light of this collaboration, and hopes that Alzheimer's treatments, tested using the clinical expertise of the ADC, will be one that comes to fruition.

Joseph Rogers

Joseph Rogers can remember when the Sun Health Research Institute (SHRI) occupied a vacant office building with himself as a staff of one. His first lab bench was a folding plastic table. Thanks in large part to his talent, perseverance, and, he is quick to point out, "the generosity of the people of Sun City," the institute has grown by leaps and bounds, and is today an internationally recognized facility.

The institute, for which he serves as president and senior scientist, now encompasses a 50,000-square-foot research building and a separate administration building/library. In addition to hosting some of the nation's top neuroscientists, the institute brings in more Alzheimer's grant money and generates more Alzheimer's publications than all the other Arizona institutions combined.

Rogers and his team were the first to implicate inflammation in the brain as a contributor to Alzheimer's, but that was just the beginning. Rogers feels that there is "no end" to the number of related scientific questions that he and his colleagues can uncover; they are "wildly excited that technology that didn't exist five years ago is now in their backyard," and available to aid in answering some of those questions.

In addition to this array of questions, the SHRI brings to the collaboration its remarkable brain donation center, where they have already enrolled more than 2,000 people. Each volunteer is evaluated annually by staff clinicians, so there is a wealth of information to go with the final brain tissue samples. Most people enrolled at the center live in Sun City, close to the SHRI facilities, a fact that has helped to improve organ recovery time and preservation after death. The SHRI also has a 24-hour answering service, its own transport, and an always- on-call rotating autopsy team that allows brain tissue to be preserved faster than anywhere else in the world: two hours, 45 minutes from death to preserved tissue. These tissue samples, along with those from brain banks at Washington University at St. Louis, Duke University, and the University of Washington, provide the starting material for the gene analysis studies.

Another major focus of SHRI has been developing methods for keeping some of the neurons from autopsy alive in a test tube for further study. These neuron cultures provide a way to functionally validate the gene expression experiments. The expression of a gene in cultured cells can be selectively "turned off," so that the corresponding protein is not made. Researchers first use the expression profiling to get a list of genes with increased expression, for example, in cells with tangles. Each of these genes can then be turned off one at a time in order to see whether each of them has an effect on the presence of tangles in the cultured cells.

Rogers believes that this type of approach—starting with expertise in an area, performing gene expression studies, and then following up with validation in the laboratory—could "short cut by half the time needed to understand these diseases and develop new cures." Already, the studies have revealed about 20 genes whose expression is altered in tangle-containing neurons and that may be causative for the tangle formation.

Rogers sees a "very bright light" at the end of the tunnel of this devastating disease. "I hope to be out of a job in seven years," he says.