Using this new technique in mice, the team discovered that the relationship between levels of a key molecule involved in Alzheimer's disease, amyloid-beta (ABeta), in interstitial fluid and cerebrospinal fluid changes as the disease progresses. Cerebrospinal fluid -- the fluid that cushions and surrounds the brain - is a main focus in efforts to diagnose and possibly treat Alzheimer's disease.
"The most exciting part of this study is that we now have a way to measure a pool of ABeta that previously could not be evaluated," says John R. Cirrito, a graduate student in neuroscience. "Using this new approach, we were able to identify another difference between young mice that have not yet developed Alzheimer's-like changes and those that have developed Alzheimer's-like brain changes, which provides a new opportunity to explore the development of this disease."
Cirrito is first author of the study, which will be published in the Oct. 1 issue of The Journal of Neuroscience. The principal investigator is David M. Holtzman, M.D., the Andrew B. and Gretchen P. Jones Professor of Neurology and head of the Department of Neurology, the Charlotte and Paul Hagemann Professor of Neurology and a professor of molecular biology and pharmacology. Collaborators at Lilly include Patrick May, Ph.D., Ronald DeMattos, Ph.D., Kelly Bales and Steven Paul, M.D.
A key step in the development of Alzheimer's disease is the formation of sticky, senile plaques in the brain, composed primarily of clumps of ABeta. Although these plaques are believed to form at least in part in the spaces between brain cells, there previously was no way to selectively extract and measure levels of ABeta in interstitial fluid.
To remove brain fluid samples for evaluation, scientists often use a technique called microdialysis, in which a miniscule tube is inserted into the part of the brain being studied. A substance, called a membrane, at the tip of the tube ensures that only freely mobile molecules are collected.
The main obstacle to studying ABeta in interstitial fluid is that the molecule is much larger than those typically measured with microdialysis. ABeta molecules also tend to stick to each other and to other particles, making it even more difficult to lure them into a tiny tube through traditional membranes. Cirrito and colleagues therefore developed a unique way to perform microdialysis, incorporating two key components: a membrane that captures larger molecules and proteins that make ABeta less sticky.
"ABeta has a lot of finicky properties, which makes it tough to capture with microdialysis," Cirrito explains. "But we put a lot of time and thought into this and finally got it to work."
Armed with this new experimental method, the team discovered some surprising things about of ABeta levels in the interstitial fluid of mice that develop Alzheimer's-type brain changes.
Two key types of ABeta are ABeta40 and ABeta42. Cirrito's team confirmed that in cerebrospinal fluid, ABeta42 levels decrease as the disease progresses, whereas ABeta40 remains unchanged. Surprisingly, they discovered a different pattern in interstitial fluid: ABeta42 remains constant while ABeta40 increases.
Moreover, levels of overall ABeta in interstitial fluid and cerebrospinal fluid were not correlated in young animals but were correlated in older, plaque-ridden mice.
"ABeta that ends up in the cerebrospinal fluid comes from interstitial fluid, so you'd expect the two compartments to communicate," says Cirrito. "Therefore, we were surprised to find that they were not correlated in young mice, and that there apparently is a shift during aging and/or during plaque development that affects how ABeta is moved between the two compartments."
Because microdialysis is performed in living animals, the team was able to take multiple interstitial fluid samples from each animal over the course of several hours. This provided the opportunity to study the breakdown and accumulation of ABeta over time, revealing new information about how the brain metabolizes and gets rid of this molecule.
The team first measured interstitial fluid ABeta levels every hour for eight hours. Then they injected the animals with a type of drug called a gamma-secretase inhibitor, which drastically decreases production of ABeta and currently is being investigated as a potential therapy for humans with the disease. Ten more interstitial fluid samples were collected over the following ten hours to measure how quickly the ABeta that had accumulated before the injection was broken down.
Comparing the rate of ABeta degradation in young and old mice, the team found that the half-life of ABeta was about twice as long in older mice. In other words, it takes about twice as long for the soluble, or mobile, pool of brain ABeta to breakdown in mice with Alzheimer's-like brain plaques than in young mice without plaques. Curiously, however, the baseline concentration of ABeta in old and young mice was not significantly different. According to the researchers, this may suggest that another, previously unidentified mechanism is involved in the development of Alzheimer's plaques.
"The difference in the elimination rate may turn out to be an extremely important finding," Holtzman says. "This suggests that once plaques form, they alter the metabolism of ABeta in the brain in a very specific way. This finding and technique should assist us in determining how other molecules that are involved in ABeta metabolism influence Alzheimer's disease as well as be a useful tool in the development of new diagnostic and treatment strategies."
Cirrito JR, May PC, O'Dell MA, Taylor JW, Parsadanian M, Cramer JW, Audia JE, Nissen JS, Bales KR, Paul SM, DeMattos RB, Holtzman DM. In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life. The Journal of Neuroscience, 23(26), Oct. 1, 2003.
Funding from the National Institutes of Health, the MetLife Foundation and Eli Lilly and Co. supported this research.
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