Amyloid β(Aβ) 1-42 fibrillation is a crucial step in the development of pathological hallmarks, such as neuritic plaques and neurofibrillary tangles, of Alzheimer’s disease (AD). In this study, we evaluated the effects of free docosahexaenoic acid (DHA), an essential brain polyunsaturated fatty acid (PUFA), on the inhibition of Aβ 1-42 fibrillation by fluorescence correlation spectroscopy (FCS), a technique capable of detecting molecular movements and interactions in solution. We also examined whether free arachidonic acid (AA), eicosapentaenoic acid (EPA), and metabolites of DHA, including neuroprotectin D1 (NPD1, 10S, 17S-dihydroxy-DHA), resolvin D1 (RvD1, 7S, 8R, 17S-trihydroxy-DHA), and didocosahexaenoyl glycerol (diDHA), affect Aβ 1-42 polymerization. The results of the FCS study reveal that DHA and AA significantly reduced the diffusion time of TAMRA (5-carboxytetramethylrhoda-mine)-Aβ 1-42 by 28% and 31%, respectively, while EPA, NPD1, RvD1, and diDHA had no effects on diffusion time. These results indicate that DHA and AA inhibited Aβ 1-42 polymerization and that their inhibitory effects occurred at the initial stage of Aβ 1-42 polymerization. This study will advance the research on PUFAs in preventing AD progression.
Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by the deposition of amyloid β (Aβ) peptides in neuritic plaques and neurofibrillar tangles in the affected brain regions [
FCS is a correlation analysis of fluctuations in the fluorescence intensity of fluorescent compounds excited by a sharply focused laser beam in a very tiny space, i.e., the so-called confocal volume. The fluorescence intensity fluctuates because of Brownian motion of the fluorescent particles. In other words, the number of particles in the confocal volume is randomly changing around the average number. This analysis gives the average number of fluorescent particles and average diffusion time when particles are passing through the tiny confocal volume. In practice, the fluorescence of dye-labeled amyloid Aβ1-42 changes because of diffusion in the confocal volume, thus the diffusion time in the presence or absence of DHA might provide greater insight into the effects of DHA on the molecular interactions of amyloid species undergoing fibrillogenesis. In addition, the effects of other PUFAs such as eicosapentaenoic acid (EPA), a precursor for DHA, and arachidonic acid (AA), the abundant n-6 PUFA in the brain, on amyloid polymerization are also unknown and thus might be studied using this technique. The DHA/AA ratio has been shown to have a significantly negative correlation with long-term memory in Aβ peptide-infused AD model rats [
The chemical structures of the compounds used in this experiments are indicated in
Aβ1-42 peptide was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at a concentration of 100 μM to produce uniform, non-aggregated Aβ and stored at −30˚C until use. On the day of use, the HFIP-dissolved amyloid was blown with N2 gas at ice cold temperature and redissolved in the assembly buffer [phosphate buffered saline (pH 7.4) containing 0.05% Tween 20].
DHA, EPA, AA, NPD1, and RvD1 dissolved in ethanol were stored at −80˚C, and diDHA dissolved in chloroform was stored at −30˚C until use. On the day of use, DHA, EPA, AA, and diDHA were mixed with assembly buffer at a final concentration of 20 μM, and NPD1 and RvD1 were mixed at a final concentration of 50 nM. Only freshly prepared DHA, EPA, AA, NPD1, RvD1, and diDHA were used.
In the present experiment, the FCS measurements were performed on a Fluoro Point Light (Olympus, Tokyo, Japan) at room temperature using the on-board 543- nm helium/neon laser at a laser power of 100 μW for excitation. TAMRA-Aβ1-42 dissolved in 1% NH4OH was stored at −30˚C. On the day of use, it was re-dissolved in assembly buffer at 1 nM, with or without DHA, EPA, AA, NPD1, RvD1, and diDHA, and quickly mixed with non-labeled Aβ1-42. Free rhodamine was used as a reference dye. The measurements were performed in a sample volume of 50 μL in a 384-well glass-bottomed microplate. The samples were sequentially and automatically loaded into the device, the optical system of which was also automatically adjusted for each measurement. Initially, the samples were subjected to FCS measurement at zero time. Afterward, the samples were incubated at 37˚C for 1 h, followed by a second reading using the Fluoro Point Light device. All experiments were performed under identical conditions, with a data acquisition time of 10 s per measurement, and measurements were repeated five times per sample. Only freshly prepared TAMRA-Aβ1-42 was used.
Results are expressed as means ± S.E. The data were analyzed by unpaired Student’s t-test and one-way ANOVA. ANOVA followed by Dunnett’s test was used for post hoc comparisons. The statistical program used was PASW Statistics 18.0 (IBM-SPSS, Inc., USA). Statistical significance was set at P < 0.05.