(182d) Detection of Active Amyloid-Beta Species Using a Quartz Crystal Microbalance

Authors: 
Moss, M. A., University of South Carolina
Kotarek, J. A., University of South Carolina


Alzheimer's disease (AD), the leading cause of dementia in the elderly, is characterized by the presence of amyloid plaques in the brain, composed primarily of the fibrillar form of the amyloid-beta protein. Amyloid-beta deposited within plaques consists principally of the 40- or 42-residue form of the peptide derived by proteolysis of cellular amyloid precursor protein (APP). Monomeric amyloid-beta self-associates to form the fibrillar A-beta that deposits within amyloid plaques. Initial aggregation of amyloid-beta monomer occurs via a rate-limiting nucleation step. Following nucleus formation, rapid growth ensues and proceeds through a soluble intermediate known as a protofibril. Two mechanisms of protofibril growth have been identified: elongation by monomer deposition and direct protofibril association. These mechanisms can be isolated in vitro by controlling monomer concentration and ionic strength.1 Although the relationship between amyloid plaques and AD is still unclear, increasing evidence suggests a role for amyloid-beta assembly in the progression of AD, and what has become known as the amyloid hypothesis continues to gain support. Consequently, the detection of active amyloid-beta species capable of assembly into protofibril intermediates or mature fibrils could be central to disease diagnosis.

In this study, a quartz crystal microbalance (QCM) is employed to detect the assembly of amyloid-beta at physiological concentrations. QCM utilizes the piezoelectric effect in quartz crystals to detect changes in bound elastic mass as a variation in the frequency of oscillation.2 Here, pre-formed biotinylated amyloid-beta aggregates are immobilized onto a Au-coated QCM surface functionalized with avidin. Subsequent addition of unlabeled amyloid-beta monomer leads to growth of immobilized protofibrils via an elongation growth mechanism. The incorporation of monomer can be detected as a change in oscillation frequency, and the quantification of elongated mass allows a relative comparison of growth potential.

Using this technique, plasma or cerebral spinal fluid isolated from AD patients may be probed selectively for the presence of amyloid-beta species capable of supporting self-assembly. QCM is able to sense mass changes in the ng/cm2 level, permitting the detection of physiological amyloid-beta concentrations, which are typically in the high picomolar range.3 Current methods for detecting physiological amyloid-beta concentrations cannot distinguish between species with different aggregation tendencies. The specific detection of amyloid-beta species capable of assembly using QCM could provide a means of early diagnosis for AD.

1 Nichols, M. R., Moss, M. A., Reed, D. K., Lin, W.-L., Mukhopadhyay, R., Hoh, J. H., and Rosenberry, T. L. (2002) Growth of beta-amyloid(1-40) protofibrils by monomer elongation and lateral association. Characterization of distinct products by light scattering and atomic force microscopy. Biochemistry 41, 6115-6127.

2 Marx, K. A. (2003) Quartz crystal microbalance: a useful tool for studying thin polymer films and complex biomolecular systems at the solution-surface interface. Biomacromolecules 4, 1099-1120.

3 Scheuner, D., Eckman, C., Jensen, M., Song, X., Citron, M., Suzuki, N., Bird, T. D., Hardy, J., Hutton, M., Kukull, W., Larson, E., Levy-Lahad, E., Viitanen, M., Peskind, E., Poorkaj, P., Schellenberg, G., Tanzi, R., Wasco, W., Lannfelt, L., Selkoe, D., and Younkin, S. (1996) Secreted amyloid beta-protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease. Nature Med 2, 864-870.