Large aggregates of the β-amyloid peptide: Honeycomb formation and stability investigated by light microscopy and light scattering in comparison with Lorenz-Mie scattering theory. A potential in vitro model system for amyloid plaques?

In aqueous medium, the β-amyloid peptide (Aβ) partly exists as large (~ 100 μm) aggregates of various forms. When examined under light microscopy, synthetic Aβ 1-42 aggregates appear mainly as flat, leaf-like, crystalline transparent structures, exhibiting from only a few to a large number of holes penetrating into the structure, producing a planar web-like or honeycomb architecture consisting of ~ 200 nm thick fibril bundles with repetitive elements (holes) of ~ 3 μm. When added to primary neuronal cultures, these structures exhibit a remarkable longevity. Direct microscopic observation shows that such aggregates are spontaneously formed during liophylization of Aβ and that they exhibit a significant stability even in the presence of organic solvents and strong denaturating agents such as urea and guanidine hydrochloride. Right-angle light scattering experiments in the presence of chaotropic agents seem to corroborate these results, when the changes in refractive index of the medium in the presence of denaturants are taken into account. The experimental results are further compared with theoretical predictions obtained from calculations using general Lorenz-Mie scattering theory. These calculations reveal important general constraints for scattering experiments employing chaotropic agents. In denaturant solutions, the sedimentation of Aβ aggregates is shown to be dependent on solvent viscosity and density changes, which offer a qualitative explanation for the observed changes in light scattering. Furthermore, staining of Aβ aggregates with thioflavine T indicates changes in dye binding in the presence of different guanidine concentrations, but no changes at different urea concentrations. In conclusion, large Aβ aggregates show structural, chemical and tinctorial properties that are similar to Aβ plaques found in the brains of Alzheimer’s disease patients, suggesting that they may constitute a simple model system for in vitro experiments. Supported by Howard Hughes Medical Institute, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro and Conselho Nacional de Desenvolvimento Científico e Technológico. stract In aqueous medium, the β-amyloid peptide (Aβ) partly exists as large (~ 100 μm) aggregates of various forms. When examined under light microscopy, synthetic Aβ 1-42 aggregates appear mainly as flat, leaf-like, crystalline transparent structures, exhibiting from only a few to a large number of holes penetrating into the structure, producing a planar web-like or honeycomb architecture consisting of ~ 200 nm thick fibril bundles with repetitive elements (holes) of ~ 3 μm. When added to primary neuronal cultures, these structures exhibit a remarkable longevity. Direct microscopic observation shows that such aggregates are spontaneously formed during liophylization of Aβ and that they exhibit a significant stability even in the presence of organic solvents and strong denaturating agents such as urea and guanidine hydrochloride. Right-angle light scattering experiments in the presence of chaotropic agents seem to corroborate these results, when the changes in refractive index of the medium in the presence of denaturants are taken into account. The experimental results are further compared with theoretical predictions obtained from calculations using general Lorenz-Mie scattering theory. These calculations reveal important general constraints for scattering experiments employing chaotropic agents. In denaturant solutions, the sedimentation of Aβ aggregates is shown to be dependent on solvent viscosity and density changes, which offer a qualitative explanation for the observed changes in light scattering. Furthermore, staining of Aβ aggregates with thioflavine T indicates changes in dye binding in the presence of different guanidine concentrations, but no changes at different urea concentrations. In conclusion, large Aβ aggregates show structural, chemical and tinctorial properties that are similar to Aβ plaques found in the brains of Alzheimer’s disease patients, suggesting that they may constitute a simple model system for in vitro experiments. Supported by Howard Hughes Medical Institute, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro and Conselho Nacional de Desenvolvimento Científico e Technológico. Origin of the honeycomb structures Aβ was pretreated with trifluoroacetic acid (TFA) under sonication and dissolved in hexafluoroisopropanol (HFIP) according to Jao et al. (1997). A drop of Aβ in HFIP was applied onto a cover slip. The left image demonstrates that the drying Aβ partially aggregates in honeycomb structures. The rim on the left side of the image is the rim of the dried droplet. One should also observe that the honeycomb structure converts into a transparent crystalline structure towards the outer rim of the dried droplet and such transparent samples are also found in lyophilized Aβ samples. The two images at the right side are transmission LM images of lyophilized Aβ1-42, as purchased from Bachem (Torrance, CA, USA), dispersed on cover slip. The structural forms of the peptide aggregates range from small sticks to large flat crystalline pieces. Theses pieces in certain cases are covered with ellipsoid holes (with axis lengths between 0.5-4 μm) in varying densities, reaching in its extreme case honeycomb like planar nets made of fibril bundles (about 200 nm thick). The image in the middle shows that also rod-like structures can be formed out of multiple fiber bundles. rigi of t e o eyco str ct res Aβ was pretreated with trifluoroacetic acid (TFA) under sonication and dissolved in hexafluoroisopropanol (HFIP) according to Jao et al. (1997). A drop of Aβ in HFIP was applied onto a cover slip. The left image demonstrates that the drying Aβ partially aggregates in honeycomb structures. The rim on the left side of the image is the rim of the dried droplet. One should also observe that the honeycomb structure converts into a transparent crystalline structure towards the outer rim of the dried droplet and such transparent samples are also found in lyophilized Aβ samples. The two images at the right side are transmission LM images of lyophilized Aβ1-42, as purchased from Bachem (Torrance, CA, USA), dispersed on cover slip. The structural forms of the peptide aggregates range from small sticks to large flat crystalline pieces. Theses pieces in certain cases are covered with ellipsoid holes (with axis lengths between 0.5-4 μm) in varying densities, reaching in its extreme case honeycomb like planar nets made of fibril bundles (about 200 nm thick). The image in the middle shows that also rod-like structures can be formed out of multiple fiber bundles. 18 8 μm 11 5 μm Solubilization of Aβ1-42 aggregates The Aβ aggregates were observed directly microscopically during treatment with different solvents. The following observations refer to changes in the aggregate structure observable by light microscopy. It shall not be excluded that small amounts of Aβ still dissolve even in cases, in which no structural changes could be detected: (1) PBS and high concentrations of urea and guanidine hydrochloride did not dissolve the aggregates. (2) TFE (pure or 50%) did not dissolve the aggregates even after multiple TFE flushing and evaporation. (3) DMSO did not dissolve the aggregates. The aggregates could be observed floating in and on top of the solution. (4) TFA dissolved the aggregates very effectively and immediately upon contact. After TFA evaporation a continuous film of Aβ could be observed on the surface of the cover slip. The effectiveness of TFA pretreatment could also be confirmed: Aggregates, which had only been partially in contact with the solvent at their outside before the TFA evaporated, did dissolve in DMSO – a process not achieved without the pretreatment. Smaller aggregates dissolved immediately in the DMSO, while the bigger aggregates dissolved to a drop of concentrated Aβ in the DMSO solution, which afterwards showed internal aggregation/disaggregation processes. (5) Formic acid dissolved the aggregates very effectively and immediately upon contact. (6) 50 mM Tris-HCl (pH 7.5) containing 2% SDS and 2mM EDTA started to dissolve the aggregates slowly. An evaporating drop of the solvent on top of the aggregates created a flow of solvent across the aggregates to the outer rim of the solvent. It carried with it dissolved particles of the aggregates in variable sizes, which partially stayed in constant circulation and partially sedimented at the outer rim of the solvent drop. Solubility profile of AD amyloid plaques Masters et al., 1985: o PBS o Urea o Guanidine hydrochloride Guanidinium thiocyanate o Ethanol o Methanol Phenol Formic acid ± 10% NaDodSO4/10% 2-mercaptoethanol Kuo et al., 2001: APP23 transgenic mice plaques: 50 mM Tris-HCl (pH 7.5) with 2% SDS and 2mM EDTA o AD plaques: 50 mM Tris-HCl (pH 7.5) with 2% SDS and 2mM EDTA Comparison of solubility profiles The AD amyloid plaques are composed of complex mixtures of Aβ (with strongly degraded Nterminal regions), glycoproteins and glycolipids, of which the latter two represent ~20 % of the total amyloid core mass (Roher et al. 1988; Roher et al. 1993a) and it has been stated that amyloid core insolubility is partially due to the presence of these ancillary molecules (Kuo et al. 2001). Comparison with the solubility profile of the Aβ 1-42 aggregates therefore allows the following conclusion: The observation that aggregates of synthetic Aβ 1-42 are stable in urea, guanidine hydrochloride, PBS, TFE and DMSO solutions, indicates that the extreme resistance of amyloid cores present in AD patients to denaturing agents and solvents can be probably understood as an inherent property of the Aβ itself. But the observed slow solubilization of the Aβ 1-42 aggregates in buffer containing SDS supports the notion that SDS insolubility of AD plaques is related to the presence of glycoproteins and/or glycolipids.