One of the cardinal pathologic features of Alzheimer's disease is the formation of both senile and amyloid plaques (basically protein deposits) in the brain. Until recently, these plaques were too small to see, so precise diagnosis of Alzheimer's disease could occur only at autopsy or with the use of cognitive testing that is influenced by many other issues in the patient's life, often making it inaccurate. This is one of many reasons that the disease is so perplexing. But the Minnesota Partnership Alzheimer's team has discovered a way to view amyloid plaques in vivo (in a living specimen), setting the stage for early diagnosis and therapeutic steps before dementia occur.

The Pathology of Alzheimer's Disease

In 2000, researchers scored a breakthrough when they found they could image amyloid plaques with Magnetic Resonance microimaging (MRI), a non–invasive imaging technique. By using a molecular probe to label the amyloid plaques in transgenic mice (those genetically engineered to develop Alzheimer's disease), scientists were able to take images of the amyloid plaques. The same technique worked to image the plaques in tissue samples from humans with the disease.

The success of using labeled probes to bind to the amyloid plaques both in vitro (in the lab) and in vivo (in the live mouse) was seen as promising, but it had certain difficulties, such as poor resolution. So scientists added a nontoxic high–contrast dye. It enhanced the plaques, greatly improving the quality of the MRI scans. This diagnostic technique allowed researchers to directly visualize amyloid plaques, track the progression of Alzheimer's and potentially monitor the effectiveness of plaque–reducing therapies.

Being able to visualize plaques is essential in verifying the feasibility of possible treatments. However, the team also needs to understand how plaques develop. The investigators examined the amount of plaque in different regions of the brain over time, and came to four significant conclusions. Their data showed that counts of individual amyloid plaques taken by MRI increase consistently with age in Alzheimer's mice, and that the MRI plaque counts correlated with the established plaque levels found in the same animals at autopsy. The scientists also discovered that plaques only 20 microns in size can be seen in mice as young as three months with MRI from outside the body and by nine months with in vivo MRI, plaques of 35 microns can be seen. For reference, size of the period at the end of this sentence is about 397 microns.

"The ability to image plaques in vivo and the other accomplishments coming out of this collaboration became possible because of the diversity and breadth of expertise that exists only in this unique partnership," says Michael Garwood, Ph.D., principal investigator from the University of Minnesota. "We were able to correlate our various MRI–based counts with the plaques in tissue sections for Alzheimer's mice over a span of ages. This technique is a major advancement and one that may be used to assess plaques over time, giving scientists a mechanism to test potential drugs, leading to prevention or treatment of the disease. Identifying the amyloid plaques early — before debilitating symptoms appear — could mean preventing the progression of Alzheimer's disease."

"This technique has a lot of potential — not just for Alzheimer's, but for many diseases in which identifying and measuring biomarkers would make a difference," says Mayo Clinic's Joseph Poduslo, Ph.D.

Crossing the Blood Brain Barrier

The complexity of this effort does not stop at imaging technology. One of the many problems researchers face in developing probes and contrast dyes is that they must be able to cross the blood brain barrier (BBB). The BBB is a semi–permeable shield; that is, it allows some materials to cross, but prevents others from crossing. In most parts of the body, the smallest blood vessels, called capillaries, are lined with endothelial cells. Endothelial tissue has small spaces between each individual cell so substances can move readily between the inside and the outside of the vessel. However, in the brain, the endothelial cells fit tightly together and substances cannot pass out of the bloodstream. The BBB protects the brain from foreign substances in the blood that may injure the brain, and "fooling" this barrier is a formidable task.

A Finely Tuned Scientific Production Line

Lining a quiet corridor in the Guggenheim Building in Rochester are half a dozen rooms where Dr. Poduslo and his team operate what is best described as a scientific production line. Two hours away in Dr. Garwood's lab at the University of Minnesota, behind a large glass window hums one of the largest, most complex MRI machines. It's hard to believe that the complex research of this team takes place in laboratories two hours apart, but in fact, the flow of the research and the interaction between team members is seamless. "We constantly talk by telephone and email all day — we probably communicate more than people who work at the same site," Says Dr. Poduslo. "We also spend every Wednesday together, alternating between Mayo and the University." Working as if their labs were doors apart, investigators and staff complement each other's expertise by conducting very specific parts of the research.

Many Moving Parts

The in vivo studies take place in Minneapolis, using a high–powered MRI, available only at the University in Minnesota. Dr. Garwood designs the pulse sequencing for the imaging that determines the length and timing of the pictures and he operates the MRI. His expertise in scientific software, graphics and imaging is critical to getting perfectly timed, accurate pictures. The mouse is positioned in a holder with monitors to control for respiration, movement, and body temperature. Sequential pictures of the mouse's brain are taken for one hour and forty minutes, using body monitors on the mouse to control for any differences that could be due to position, respiration or body temperature. Outside the MRI room, a researcher sits in front of a monitor with complex pictures and graphs that relay and record every twitch the mouse makes, so that pictures of the plaques can be taken over time when the mouse is in the exact same position. Any differences in the size of the plaques can then be attributed only to an increase in the quantity and size of the plaques over the life of the mouse, and can then be compared to the post–mortem tissue analysis.

Back in Dr. Poduslo's lab in Rochester, experts in protein analysis and synthesis are preparing proteins that can cross the BBB, and contrasts to make the proteins visible. Adding the contrast to the protein is a complex trial and error process, since contrast agents must be safe for the brain but still be formulated to attach to the plaques and make them visible. This protein molecule attaches itself to the amyloid plaques when injected into the mouse's brain, allowing investigators to compare pictures of the brain in vivo with post–mortem slides of the mouse's brain. This is when Dr. Clifford Jack steps in to lend his expertise in post–imaging analysis and as an internationally–regarded expert in Alzheimer's disease.

Next door, Thomas Wengenack and Geoffry Curran are responsible for raising, breeding and genotyping the transgenic mice that are such a critical component of this work. Wengenack does much of the post–mortem analysis on the mice, providing the expertise is surgery (a painstaking process given the minute size of a mouse's brain) and detailed analysis of the slides.

"We hope to employ the techniques developed in our collaboration to address important questions in our field, for example in investigating experimental treatment effects," notes Dr. Jack. "The three of us were collaborating well before the Minnesota Partnership was established, but with the support of the Partnership, and the enhanced funding, we have been able to do more, faster and publish sooner."

A Multi–disciplinary Team

All of these steps take experts in specialized areas of medical research including pharmokinetics (the distribution of materials throughout the brain), neurology and neuroscience, radiology, genomics, biochemistry, molecular biology, surgery and, of course, Alzheimer's disease. The level of expertise here is clear to the observer, but so is the comfortable working environment of this team. There is great mutual respect and admiration, but also ample laughter and joking to offset the intensity of their research.

"It can be daunting to think about the work that we are doing, the importance of it and the impact we could have on people's lives," notes Mayo's Karunya Kandimalla, Ph.D. "Because we all get a long so well, we're able to manage the occasional frustrations by supporting each other. We all really like each other and value our respective specialized areas. It's rare to find this atmosphere in such an intense, competitive field."

Doctors at Mayo believe that medical research is advanced far more by teamwork than by an individual working alone. The diversity and excitement of the Minnesota Partnership's Alzheimer's team is a 21st Century example of that belief.