June 4, 2004 People with Alzheimer’s disease have three to four times more antibodies to two major players in the destructive disease than their healthy counterparts, researchers at the Medical College of Georgia and Veterans Affairs Medical Center in Augusta have found.
The ability to measure these specific antibody levels could lead to a method for early diagnosis of the disease – when treatment has the most potential – and even help identify those at risk, say lead researchers Drs. Shyamala Mruthinti and Jerry J. Buccafusco of work currently published online on ScienceDirect and scheduled for publication in the September issue of Neurobiology of Aging.
The finding may also enable development of a monoclonal antibody that selectively destroys the deadly protein-receptor combination, they say.
The study of plasma samples from 33 patients with Alzheimer’s and 42 healthy people showed a similar effect on leukocytes, white blood cells that are part of the immune response: leukocytes from Alzheimer’s patients had four times the markers for amyloid-â peptide and receptor for advanced glycation end products, or RAGE.
The findings support the theory that autoimmunity and resulting inflammation play a big role in the destructive brain disease.
Previous studies have looked for and found only low levels of these antibodies in Alzheimer’s; Dr. Mruthinti, a neurobiologist at the VA and adjunct instructor of pharmacology/toxicology at MCG, wondered if the experiment design was to blame and designed a more specific approach that yielded results.
"This is one of the most exciting papers I have ever participated in," says Dr. Buccafusco, pharmacologist and director of the MCG Alzheimer’s Research Center. "If it turns out that the assay is a confirmatory assay for Alzheimer’s disease, it would be a very, very useful assay to begin with. But if it has a predictive value as well, it would be more outstanding, obviously." Currently there is no definitive diagnostic test for Alzheimer’s; a definitive diagnosis is made on autopsy.
Amyloid-â peptide is a protein fragment routinely found circulating in the bloodstream that is harmless as long as it stays there. The same can be said for RAGE peptide, another natural human product that actually can help attract cellular garbage in the bloodstream, such as beta amyloid, and remove it from the body. Apart, neither attracts much attention from the immune system. But when RAGE in the brain attracts beta amyloid, they combine to form a complex capable of generating a stronger immune response.
RAGE was discovered by Dr. David M. Stern, dean of the MCG School of Medicine, while he headed the Center for Vascular and Lung Pathobiology and the Juvenile Diabetes Research Center at the College of Physicians & Surgeons of Columbia University in New York. He first found RAGE was a binding receptor for sugar-modified proteins that accumulate in the blood vessels of diabetes; he and colleague Dr. Shi Du Yan published in Nature in 1996 their finding that in the brain, RAGE also binds with amyloid-â peptide in Alzheimer’s. "I am delighted that Drs. Mruthinti and Buccafusco are pushing their work in Alzheimer’s disease forward along a line of inquiry that involves RAGE," Dr. Stern says. "Their findings are both intriguing and unexpected."
"We have shown that advanced glycation end products in the presence of beta amyloid enhance the expression of specific cellular receptors such as RAGE that are known to bind the toxic form of amyloid," Dr. Mruthinti says.
She and Dr. Buccafusco began dissecting the relationship between RAGE and beta amyloid after she immunized laboratory mice with advanced glycation end products in pursuit of a monoclonal antibody that might be a diagnostic marker for Alzheimer’s.
The researchers expected and documented that the mice made antibodies to advanced glycation end products. But in studies published in 2003 in the Journal of Molecular Neuroscience, they were excited to find the mice also made antibodies to RAGE and beta amyloid, which were locked together in a deadly complex that was getting the attention of the immune system.
"It seems that when the mice were immunized, this enhanced the presence of circulating RAGE peptide which, in turn, formed the highly immunogenic complex with beta amyloid protein," Dr. Mruthinti says. "So the antibodies they measured may have been produced in response to the protein complex rather than to the original proteins." The investigators have found evidence of such RAGE-beta amyloid complexes in the blood of Alzheimer’s patients.
"We think the advanced glycation end product causes an increase in cell-surface expression of amyloid-â binding sites, which are RAGE and others, that cause all this amyloid to come and bind to the cell which is not healthy for the cell," Dr. Buccafusco says.
"It stirs up a cyclic, inflammatory event," Dr. Mruthinti says of the deadly cycle set in increasing motion.
Activation of the immune system by the resulting complex has the Augusta researchers believing, as others do, that there is an autoimmune component to the disease. But when she purified out antibodies from the plasma – instead of just looking at intact plasma as other have – and exposed them to specific affinity columns designed to attract antibodies to beta amyloid and RAGE, the researchers were able to count antibodies specific for the two and document the increase in both that occurs in Alzheimer’s. They also found a linear correlation: when amyloid-â antibodies were high, so were RAGE antibodies, probably because they are binding to the same complex, Dr. Buccafusco says.
"What we want to do is find patients who have the propensity of becoming Alzheimer’s patients," he says. "That is where a simple, early diagnostic test would come in handy. If you could tell someone they have a high chance of becoming an Alzheimer’s patient in the next five to 10 years, then you can keep an eye on them and intervene early in the disease when it will do the most good." The researchers’ study already has yielded healthy individuals with relatively high levels of the important antibodies to follow to see if Alzheimer’s develops. They also will work with another group of researchers studying mild cognitive impairment – thought to be a precursor disease – using the new assay to tests its potential as an early diagnostic tool. Meanwhile, Dr. Mruthinti is working toward developing the monoclonal antibody against the destructive complex. “If I can make a human antibody against the complex that can be used a therapy, that would be my dream,” she says.
The studies were made possible by contributions of blood by Alzheimer’s patients as well as healthy individuals to MCG’s Neurological Diseases Databank Repository. For more information about the repository, visit http://www.mcg.edu/centers/alz/.
Other co-authors include MCG neuroscientist William D. Hill, biostatistician Jennifer L. Waller and geriatrician Thomas W. Jackson as well as Rosann F. Schade, study coordinator the Neurological Diseases Databank Repository and neurologist Edward Y. Zamrini from the University of Alabama at Birmingham.
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