An antibody sequesters a protein that interferes with neural functionand could hold a key to preventing Alzheimers.
Research advances such as this have brought us to
the exciting threshold of testing strategies that, if they are effective
in humans, may arrest or even prevent Alzheimer's disease.
OF ALL THE HURDLES that block the way to understanding Alzheimers diseasethe complexity of brain metabolism, the possibility that more than one mechanism is at work, and othersone of the highest has been that the disease does its harm behind the blood/brain barrier.
That barrier, the brains protective shield, confounds observations and makes it difficult to deliver agents that might have an effect. The barrier acts as a filter, screening out large molecules and regulating the flow of small proteins, such as Amyloid beta. Many also think that Amyloid betareferred to as Abis the building block for plaques that accumulate in the brains of Alzheimers patients. Many researchers believe that Ab disrupts memory, dislocates thought processes and eventually destroys identity.
What may turn out to be the leap that clears the hurdle comes from researchers in the laboratory of David M. Holtzman, MD, the Paul and Charlotte Hagemann Professor of Neurology. Holtzman and his colleagues devised an elegant and uncommonly linear series of experiments in mice showing that it does not appear to be necessary to cross the blood/brain barrier in order to have an effect on the brain side of the equation. The work points enticingly at potential therapies for the disease.
"Amyloid beta is made by almost every cell in the body, but by far the highest production is by nerve cells in the brain," Holtzman says. "Weve shown that there are equilibriums in the concentration of Amyloid beta in the brain, in the cerebrospinal fluid (CSF), and in the plasma. And we can influence those equilibriums."
The course of the investigation began unexpectedly when Ron DeMattos, PhD, instructor in neurology and a researcher in Holtzmans lab, set out to examine the relationship between Ab and a molecule that binds to it. He was looking for the trigger that transforms Ab from its normal, apparently harmless form to the misfolded type that clumps together to create plaques.
To understand the work, DeMattos says it is important to think of proteins as bound together in complexes, because "thats what they do. In physiology, we deal with complexes." DeMattos was using the antibody m266, an antibody that binds to Ab and is therefore a tool for tracking down the protein, to find what it was binding to. He observed that the m266 he used as a probe not only bound to Ab but also prevented it from binding to other proteins and apparently captured for itself all the Ab that was available. In the words of Holtzman and DeMattos, m266 had "sequestered" all of the Ab in the solution.
That observation made, DeMattos and Holtzman set to work in earnest, with the help of colleagues Maia Parsadanian, Mark ODell, Eric Foss, Kelly Bales and Steven Paul. First, they confirmed the affinity of Ab for m266 in a benchtop experiment (see sidebar). Then they turned their attention to genetically altered mice that overproduce Ab and therefore develop Alzheimers disease-like changes in their brains.
They injected m266 or a control agent into the blood of two groups of the Alzheimers mice, then measured to see if the antibody had, in fact, sequestered Ab there. They discovered that in the m266-treated mice, they could find almost no Ab in the blood that was not bound to the antibody: It all had been sequestered. The amount of Amyloid beta that had been captured by m266 was about 1,000 times higher than what they expected to find, even in these mice designed to develop Alzheimers disease.
What was the origin of this excess of the potentially dangerous Ab? Was m266 in the blood a "sink" that altered the equilibrium of Ab in the various compartments in which it is presentthe brain, the cerebrospinal fluid, the blood? To answer those questions, the researchers first had to devise a new method of extracting cerebrospinal fluid from the mice. Such small animals produce only tiny amounts of CSF, and procedures are delicate.
New technique in hand, they again injected mice with m266 and were able to record a subsequent change in concentration of free Ab in the CSF. That the change they saw was an increase, a counterintuitive result, is not as important as the news that the presence of m266 in the blood effected a change on the other side of the blood/brain barrier, Holtzman says. Why Ab increased transiently is now under investigation, and the early thinking is that over a longer period, the concentration will indeed go down.
The hypothesis of m266 as a sink that draws Ab out of equilibrium was further demonstrated when the researchers loaded the brains of their subject mice with additional Ab, then injected the m266 antibody or a control agent into the blood. The results: With m266, a massive accumulation of Ab was measured in the blood, as much as a 365-fold increase after three hours, compared to mice that received no m266.
And, DeMattos says, the effect continues. Over 96 hours, the m266-treated mice showed more than 1,000 times as much Ab sequestered in the blood as their untreated counterparts.
But all of this work was preliminary; the big question came next. The investigators most wanted to know if increasing the presence of m266 in the blood could prevent or reduce the deposition of the sticky, thought-disrupting form of Ab in the brain.
Over five months, 14 Alzheimers-model mice were administered saline solution. A second group of 13 received a control antibody. And another group of 14 was treated with m266. When the mice were examined at nine months of age, the brains of 11 of the 27 mice in the two control groups were seriously riddled with Ab plaques. Among the 14 m266-treated mice, however, only one had the same level of plaque deposition.
Holtzman says that the labs most important result is the demonstration that brain metabolism of Ab can be altered from the blood side of the blood/brain barrier, that equilibrium can be influenced. Commenting on the work, John C. Morris, MD, Friedman Professor of Neurology and co-director of the Alzheimers Disease Research Center, says "Drs. DeMattos and Holtzman have worked out an elegant method to drastically reduce the amount of amyloid in the brain, which is thought to initiate the brain damage associated with Alzheimers." Holtzman and his colleagues now are looking for the mechanism by which the Ab is transported across the blood/brain barrier.
With m266s ability to influence Ab equilibrium, the possibilities for treating Alzheimers disease brighten considerably. "Ab can misfold under certain conditions, and everybody has the potential to get this disease," Holtzman says. But an agent that sequesters what many believe to be the culprit, prevents it from accumulating in its dangerous form, and then clears it from the system sounds like a therapy waiting only for doses to be established. And Holtzman and DeMattos say that m266 could form the basis for a therapeutic agent.
"The technology to humanize mouse antibodies is well-tested and safe," DeMattos says. Though no one yet knows what the function of normal Ab is, he is confident that dosages could be established that preserve normal function but eliminate the excess required for plaque-building. The direct application of m266 as a therapeutic medicine faces some difficulties: Holtzman says that such a drug probably would have to be administered every few weeks. "Cost might be a factor, but it certainly is possible," he says.
Another approach might be to find a similar agent with comparable effects, since Ab is known to bind to other, abundant elements, such as lipoproteins, which medical science knows how to manipulate effectively.
Says Morris: "Research advances such as this have brought us to the exciting threshold of testing strategies that, if they are effective in humans, may arrest or even prevent Alzheimers disease."
In addition to pondering preventative strategies, the researchers have begun investigating whether m266 might also reverse existing Alzheimers disease pathology. "Plaques of Ab in the brain are probably not inert," Holtzman says. Therefore, they may be accessible to treatment. Though he has no data yet to show that existing plaques can be reduced or eliminated, his lab has begun to assess the idea. In addition, his laboratory, in collaboration with Eli Lilly and Company, continues to explore the potential of using anti- Ab antibodies to develop new diagnostic and treatment options for Alzheimers disease.
Editors note: This research was performed in collaboration