School of Medicine researchers in collaboration with researchers at Eli Lilly and Co. have developed a new technique that dynamically studies proteins in the fluid between brain cells, called interstitial fluid, known to be related to Alzheimer’s disease.
Using this new technique in mice, the team discovered the relationship between levels of a key molecule involved in Alzheimer’s disease, called amyloid-beta (ABeta), changes in interstitial fluid and cerebrospinal fluid as the disease progresses. Cerebrospinal fluid, the fluid that cushions and surrounds the brain, is a main focus in diagnosing and possibly treating Alzheimer’s disease.
David M. Holtzman
“We now have a way to measure a pool of ABeta that previously could not be evaluated,” said graduate student John R. Cirrito. “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, 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.
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.
The main obstacle to studying ABeta in interstitial fluid is that the molecule is larger than those typically measured with microdialysis. The ABeta molecules also tend to be sticky, making the process even more difficult.
John R. Cirrito
Cirrito and his 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.
Armed with this new method, the 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.
“ABeta that ends up in the cerebrospinal fluid comes from interstitial fluid, so you’d expect the two compartments to communicate,” Cirrito said. “We were surprised to find they were not correlated in young mice. There apparently is a shift during aging and/or during plaque development that affects how ABeta is moved between the two compartments because levels of ABeta do correlate in older, plaque-ridden mice.”
Because microdialysis is performed in living animals, the team took multiple samples from each animal to study the breakdown and accumulation of ABeta over time.
The team first measured interstitial fluid ABeta levels every hour for eight hours.
The researchers then injected the animals with a 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 10 hours to measure how quickly the ABeta that had accumulated before the injection was broken down.
The team found it takes about twice as long for the soluble pool of brain ABeta to breakdown in mice with Alzheimer’s-like brain plaques than in young mice without plaques.
However, the baseline concentration of ABeta in old and young mice was not significantly different, which 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 said. “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 involved in ABeta metabolism influence Alzheimer’s disease as well as be useful in developing new diagnostic and treatment strategies.”