By John R. Cirrito, Ph.D., and David M. Holtzman, M.D.
Microdialysis Core Facility, Department of Neurology, Hope Center for Neurological Diseases, Washington University, St. Louis, MO
A major contributing factor in Alzheimer’s disease is the elevation of a protein called amyloid-β, or Aβ. Since high levels of Aβ play a role in the disease, many research groups around the world are developing therapies designed to lower the levels of this protein. With the help of Cure Alzheimer’s Fund, our group initiated an ambitious project to discover new drugs that reduce Aβ levels. Our approach is to test drugs in living animals (mice). While this process is time-consuming, it enables us to discover entirely new classes of compounds that traditional drug screening methods typically might overlook.
In Alzheimer’s disease, Aβ accumulates as amyloid plaques in the space between nerve cells. Several years ago, our group developed a technique called microdialysis to measure Aβ within the space immediately adjacent to nerve cells in living mice. Microdialysis has been used in the study of the brain for more than 20 years to measure small molecules; however, it is rarely used for such large molecules as proteins and had never been used to detect Aβ. This procedure requires us to implant a small microdialysis probe in the brain of an anesthetized mouse. At the tip of this probe is a semi-permeable membrane that allows brain Aβ to enter and be collected over time. We test the solution inside the probe every 60 minutes to determine Aβ levels. Once the probe is implanted into the brain, the animal is allowed to wake up and is housed in a specialized cage that permits him to move around, eat and drink, all while Aβ is being collected from the brain. Because the mouse is alive during our procedure, the changes in Aβ levels are likely to be very relevant to both normal and diseased processes in the brain.
The microdialysis probe has several uses. The probe not only enables us to extract Aβ from the brain, but also can be used to deliver drugs directly to the mouse brain in which Aβ is being measured. By adding drugs to the inside of the probe, we can treat and assess the living brain. Each mouse receives several drugs over the course of a three- to four-day study. One drug is given to each mouse for several hours and then the drug is removed for several hours before the next drug is given. This enables us to test several drugs with each mouse, thus allowing us to use mice much more efficiently and to screen compounds more quickly. Mice do not develop Alzheimer’s disease exactly as humans do. Our mice, however, have been genetically engineered to contain the human type of Aβ that accumulates in Alzheimer’s disease. At young ages (up to four months old), these transgenic mice do not contain any signs of Alzheimer’s-like changes. “Transgenic” just means that extra genes have been inserted into the mouse’s DNA. At around six months of age, however, the transgenic mice develop amyloid plaques that are remarkably similar to plaques found in human Alzheimer’s disease patients. While these types of mice are not perfect models of Alzheimer’s disease, they have proven to be excellent models for the study of the pathways that regulate levels of Aβ in the brain over time.
Because we can screen only a limited number of compounds within a several year period, we chose a library of compounds that are known to be “pharmacologically active.” This means that each drug is known to affect a specific target in the body; however, those targets are not necessarily related to Alzheimer’s disease (as far as is known). By using this pharmacologically active library, we are testing pathways that in some cases have never been implicated in Alzheimer’s disease. Our group recently discovered that normal brain activity causes Aβ to be produced. More than half of the drugs in this library affect brain activity, which increases the likelihood of finding compounds that affect Aβ levels. We are finding that some compounds have no effect on Aβ levels while other compounds increase Aβ levels. Most importantly, we already are finding compounds that lower Aβ levels in mice. These compounds in particular have important therapeutic implications. This project is exciting for us because it discovers new ways to lower Aβ, which we hope will allow us to treat the disease in the future. We think this project also is giving us new insights into the fundamental processes that contribute to Alzheimer’s disease.
There are few labs around the world performing this Aβ microdialysis technique. Many groups, however, have expressed interest in using this technology to test their own set of compounds for their ability to reduce Aβ. Consequently, we have opened our Microdialysis Core Facility to both academic and corporate groups that have novel compounds likely to influence Aβ levels. We hope this service will help other groups develop drugs to combat Alzheimer’s disease. We already have started working with several pharmaceutical companies as well as collaborating with academic labs.One approach to drug discovery is to use a minimalistic experimental system to test many thousands of potential drugs that could affect a very specific therapeutic target. Many research groups are using these systems to target known pathways that produce or eliminate Aβ, such as what are called secretase inhibitors or protease enhancers. In contrast, our approach uses the most complex system possible, an awake, behaving mouse, in order to test a small number of diverse compounds that target pathways that have been largely untested in Alzheimer’s disease. We think our method offers an opportunity to identify entirely new targets for therapeutic intervention. Though the Microdialysis Core Facility is still young, we already are discovering new leads we hope can be used to treat Alzheimer’s disease.A microdialysis probe is implanted in the brain of a living mouse. This probe enables us to collect and measure Aβ from an awake, behaving mouse in real-time. The probe also allows us to deliver drugs directly to the brain.