Familial Alzheimer's Disease Mutations within the Amyloid Precursor Protein Alter the Aggregation and Conformation of the Amyloid-β Peptide

Abstract

Most cases of Alzheimer's disease (AD) are sporadic, but a small percentage of AD cases, called familial AD (FAD), are associated with mutations in presenilin 1, presenilin 2, or the amyloid precursor protein. Amyloid precursor protein mutations falling within the amyloid-β (Aβ) sequence lead to a wide range of disease phenotypes. There is increasing evidence that distinct amyloid structures distinguished by amyloid conformation-dependent monoclonal antibodies have similarly distinct roles in pathology. It is possible that this phenotypic diversity of FAD associated with mutations within the Aβ sequence is due to differences in the conformations adopted by mutant Aβ peptides, but the effects of FAD mutations on aggregation kinetics and conformational and morphological changes of the Aβ peptide are poorly defined. To gain more insight into this possibility, we therefore investigated the effects of 11 FAD mutations on the aggregation kinetics of Aβ, as well as its ability to form distinct conformations recognized by a panel of amyloid conformation-specific monoclonal antibodies. We found that most FAD mutations increased the rate of aggregation of Aβ. The FAD mutations also led to the adoption of alternative amyloid conformations distinguished by monoclonal antibodies and resulted in the formation of distinct aggregate morphologies as determined by transmission electron microscopy. In addition, several of the mutant peptides displayed a large reduction in thioflavin T fluorescence, despite forming abundant fibrils indicating that thioflavin T is a probe of conformational polymorphisms rather than a reliable indicator of fibrillization. Taken together, these results indicate that FAD mutations falling within the Aβ sequence lead to dramatic changes in aggregation kinetics and influence the ability of Aβ to form immunologically and morphologically distinct amyloid structures.

Keywords: Alzheimer disease; aggregation; amyloid-β (Aβ); monoclonal antibody; peptide conformation.

FIGURE 1.

Aggregation kinetics of WT and FAD mutant Aβ peptides. a, ThT fluorescence profiles for WT Aβ and 10 Aβ variants containing FAD mutations. b, expanded view of the ThT fluorescence data for selected variants with maximum fluorescence values similar to that of WT Aβ. The peptides were aggregated under condition A (NaOH/phosphate buffer method). ThT fluorescence intensities for each time point were obtained in triplicate and are presented as the mean of the three values. This experiment was performed in duplicate with similar results.

FIGURE 2.

Morphologies of WT and FAD mutant Aβ peptides. TEM images of the aggregates formed by WT Aβ40 and 11 Aβ40 FAD variants under condition A (resuspension in NaOH followed by dilution in phosphate buffer) after 10 days are shown. This experiment was performed twice, with similar results, and representative images from three technical replicates per peptide are displayed. Scale bar, 10 nm and is the same for all panels.

FIGURE 3.

Immunological analysis of aggregation by dot blotting at representative time points. Dot blotting results from WT Aβ and 11 Aβ variants containing FAD mutations aggregated using condition A (NaOH/phosphate buffer method) (a), condition B (HFIP/water method) (b), and condition c (NaOH/HEPES/NaCl method) (c). The peptides were aggregated over a 10-day time course, and the selected time points were probed for their immunological reactivity with the indicated antibodies. This figure displays only the time 0 and the 3- and 10-day time points of the aggregation reactions. The full data are presented in Fig. 4. These experiments were performed in duplicate with similar results.

FIGURE 4.

Immunological analysis of aggregation by dot blotting. Full set of dot blotting data from WT Aβ and 11 Aβ variants containing FAD mutations aggregated using condition A (NaOH/phosphate buffer method) (a), condition B (HFIP/water method) (b), and condition c (NaOH/HEPES/NaCl method) (c). The peptides were aggregated over a 10-day time course, and the selected time points were probed for their immunological reactivity with the indicated antibodies. Fig. 3 is an abbreviated version of this figure included to enable easier interpretation of the data. These experiments were performed in duplicate with similar results.

FIGURE 5.

Western blotting analysis of aggregation. Western blotting results from WT Aβ and 11 Aβ variants containing FAD mutations aggregated under conditions A, B, and C (a–c, respectively). Samples from time 0 and the 3- and 10-day time points were subjected to SDS-PAGE and probed with the widely reacting representative antibody mOC87. These experiments were performed in duplicate with similar results.

FIGURE 6.

Relationship between location of FAD mutation and antibody epitope and reactivity. Dot blotting results for Aβ40 and 11 Aβ variants associated with FAD aggregated under conditions A–C probed with antibody mOC98 (a) and mOC116 (b), along with epitope mapping data for each of the antibodies. Blue depicts the results of a SPOT epitope mapping experiment. Each of the blue panels depicts 40 spots containing peptides C-terminally bound to a cellulose membrane. The peptides are consecutive 10-amino acid-long segments of the Aβ sequence beginning at position −3 (top left corner). The blue color indicates antibody reactivity with a particular spot. The interpretation of the epitope mapping study is to the right of the results, with the apparent epitope highlighted in red. In this assay, a large number of consecutive reactive spots indicates a shorter epitope, as there are a smaller number of amino acids common to the reactive spots. Conversely, a small number of consecutive reactive spots indicates a longer epitope, as the longer epitope would only be contained within a smaller number of spots.