St. Louis, April 28, 1998 -- Since 1992, scientists have known that people with a particular form of the protein apoE are at increased risk for developing Alzheimer's disease. They have been frustrated, however, because there has been no way to find out how the biological effects of this variant, called apoE4, differ from those of apoE3, the most common apoE in humans. Researchers at Washington University School of Medicine in St. Louis now have offered a solution by generating mice that make only human apoE3 or human apoE4. Their experiments with these mice show that apoE3 promotes nerve cell outgrowth, whereas apoE4 does not.
Most people have either two apoE3 genes, two apoE4s or one of each. The apoE2 gene is much rarer. Inheriting two apoE4s seems to be the worst luck of the draw. As well as lowering the age at which Alzheimer's disease develops, this permutation appears to speed the onset of Parkinson's disease and bodes a poor neurological outcome after cardiac bypass or cerebral hemorrhage.
"Our thinking is that apoE3 is much more effective at promoting regrowth of nerve cell extensions after injury," says David M. Holtzman, M.D., assistant professor of neurology and molecular biology & pharmacology. "Or it might be protective in the setting of brain diseases by preventing loss of connections between neurons."
Holtzman is chief author of a paper in the May 1 issue of Journal of Neuroscience. Research associate Yuling Sun, is first author.
To generate the experimental animals, Sun inserted human apoE3 or apoE4 genes into mice. Through other genetic manipulations, she made the mice express the human gene only in astrocytes, the housekeeping cells that normally make apoE in the brain. The transgenic animals then were crossed with mice that lacked the genes for mouse apoE. Eventually, the researchers obtained litters of two types. One contained pups that made no apoE plus pups that made only human apoE3. The other contained pups that made no apoE plus pups that made only human apoE4.
The researchers collected astrocytes from the newborn mice and placed them in culture dishes. When the astrocytes had converged, they covered them with neurons from the hippocampus of normal embryonic mice, mimicking the community of astrocytes and neurons that exists in the brain. The hippocampus is a seahorse-shaped structure associated with memory and learning, functions that are compromised in Alzheimer's disease, where connections between nerve cells disappear.
The astrocytes secreted particles made of cholesterol, lipid and apoE in a form that is likely to occur in the brain. When these particles contained apoE3, the neurons developed long arms or neurites. Growing neurites reach out to other nerve cells like telephone wires, laying down or repairing the infrastructure for cell-to-cell communication.
When the particles contained apoE4, the neurites were no longer than those from the neurons with no access to apoE. Measuring them in each dish, the researchers found that the total length of neurites exposed to apoE3 was about 50 percent greater than those exposed to apoE4 or no apoE.
"So lipoprotein particles that may mimic those secreted by astrocytes in the brain have different effects on neuronal structure according to whether they contain apoE3 or apoE4," Holtzman says. "One could speculate that such an effect on structural plasticity might provide a common mechanism for the disadvantageous effects of apoE4 in Alzheimer's disease, Parkinson's disease and recovery from bypass surgery and cerebral hemorrhage."
The researchers will explore this idea further. The apoE3 and apoE4 mice also will be invaluable for a wide range of other experiments, Holtzman predicts. For example, they should be useful in determining how the different forms of apoE in brain affect the buildup of amyloid plaques in Alzheimer's disease. "There is a great deal of interest in the mechanism," Holtzman says. "These mice will provide a powerful way of exploring the role of human apoE isoforms produced in the brain and their influence on brain diseases."
Sun Y, Wu S, Bu G, Onifade MK, Patel SN, LaDu MJ, Fagan AM, Holtzman DM. Glial Fibrillary Acidic Protein-Apolipoprotein E (apoE) Transgenic Mice: Astrocyte-Specific Expression and Differing Biological Effects of Astrocyte-Secreted apoE3 and apoE4 Lipoproteins. Journal of Neuroscience, 18(9), 3261-3272, May 1, 1998.
Grants from the National Institutes of Health, Alzheimer's Association and a Paul Beeson Physician Faculty Scholar Award from the American Federation for Aging Research supported these studies.