Contact: Lauren Ward
412-268-7761
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For immediate release:
January 19, 2004
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Carnegie Mellon Scientists Use Atomic Force Microscopy to Discover Effects of Experimental Alzheimer’s Drugs on Plaque Formation
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AFM images taken on the fifth day of incubation clearly show the impact of antibodies binding to the different portions of the peptide on Ab aggregation. While Ab alone forms numerous extended fibrils (top), in the presence of m3D6 which binds to the N-terminus the fibrils are less numerous and their periphery is surrounded by a "halo" of antibodies (middle). In great contrast, no fibrils are formed in the presence of m266 which binds to the central portion of the peptide, and only compact, globular aggregates are observed (bottom). The ability of AFM to quickly assess the extent of Ab fibrillogenesis may greatly aid the efforts aimed at the development of agents which would suppress it or even reverse its progress. Such agents could find future therapeutic use in the treatment of Alzheimer's disease. |
PITTSBURGH—Scientists at Carnegie Mellon University have used atomic force microscopy (AFM) to shed light on molecular scale processes underlying the formation of insoluble plaques associated with Alzheimer’s Disease. Results of this work suggest that AFM could lead to a better understanding of the disease process and help guide the search for new diagnostic and treatment approaches. The report will be published in Volume 335, Jan. 23 issue of the Journal of Molecular Biology and the abstract appears online at www.sciencedirect.com/web-editions/journal/00222836.
Supported by the National Institutes of Health, the research was done in collaboration with scientists at Eli Lilly and Company and the Washington University School of Medicine.
“Because AFM provides three-dimensional topographical information at the nanoscale, it could prove important in assessing the potential usefulness of molecules like antibodies to effectively inhibit protein aggregation associated with Alzheimer's Disease,” said Tomasz Kowalewski, Ph.D., assistant professor of chemistry at Carnegie Mellon and senior author on the paper.
Alzheimer’s Disease belongs to a class of disorders called conformational diseases, which are caused by changes in a protein’s physical state. Another widely known example is prion-based “Mad Cow” disease. Tools used to understand these disorders are targeted primarily to biochemical processes, noted Kowalewski, who works at the Mellon College of Science. “Because AFM probes the physical state of proteins, it could really assist in understanding conformational diseases, which traditionally have been difficult to fight,” said Kowalewski.
A hallmark of Alzheimer’s Disease is the presence of insoluble plaques in the brains of disease victims. These plaques are made of beta amyloid (A ), a peptide derived from a precursor protein found in cell membranes. Once cleaved from its parent protein, the A peptide readily aggregates into fibrils that are rich in insoluble stacked structures called beta sheets. These fibrils then congeal into tangled plaques. For the past few years, scientists in the field have been focusing considerable efforts on developing molecules that would inhibit A aggregation or perhaps even break up existing plaques. In this quest, they have recently turned their attention to antibodies that bind to different portions of the A peptide.
In their study, Kowalewski and a graduate student, Justin Legleiter, incubated the A peptide in solution with two different monoclonal antibodies: m3D6, which binds near one end of the peptide called the amino terminus, and m266.2, which binds to the peptide’s central portion. Over several days, the researchers placed drops of the sample solutions on mica sheets and observed the degree of protein aggregation using AFM. The m266.2 antibody proved much better than m3D6 at preventing the formation of amyloid fibrils.
“Interestingly, we found that both antibodies interfere with the formation of protofibrils, or fibril precursors,” noted Kowalewski. “In solutions of A alone, numerous protofibrils were present under our experimental conditions as early as the third day, whereas in the presence of m3D6 they grew at a slower rate. We found that m266.2 completely inhibited protofibril formation.”
The scientists believe that the two antibodies differ in their ability to inhibit fibril formation due to the way they bind to the A peptide. Binding at one end of A, m3D6 does not inhibit the formation of extended beta sheets, which are the major structural feature of mature fibrils. Because m266.2 binds to the center of A, it blocks the formation of these extended beta sheets, the investigators surmised.
In AFM, a tiny lever ending with an ultra-sharp tip is scanned across a surface from side-to-side and top-to-bottom, much as a cursor moves across a computer screen. A laser beam reflected off the lever’s end monitors its vertical motion. Perturbation of the lever motion by nanoscale features of a sample’s surface topography is used to reconstruct a detailed three-dimensional map of the surface. In the study, the research team used custom-written software to process and to analyze their AFM images.
AFM is an appealing tool, according to Kowalewski, because it provides three-dimensional nanoscale images and, unlike some other techniques, it does not require specialized preparation or contrasting agents that could introduce artifacts. In addition, it allows to study the samples in liquids, under nearly physiological conditions, according to Kowalewski, who is now pursuing this kind of AFM studies to understand the interaction of antibodies and lipoproteins, with the A peptide.
Collaborating investigators include Dan L. Czilli, Bruce Gitter and Ronald B. DeMattos (Eli Lilly and Company) and David M. Holtzman (Washington University).
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For more information, see:
 Tomasz Kowalewski
Journal of Molecular Biology
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