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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 26, 2005 as doi:10.1096/fj.04-3137fje. |
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,¶
* Laboratori de Recerca en Nanobioenginyeria, Parc Científic de Barcelona (PCB), Universitat de Barcelona (UB), Spain;
Departament de Química Física, UB;
Institut de Recerca Biomèdica de Barcelona, PCB;
Facultat de Ciències de la Salut, Universitat Internacional de Catalunya, Barcelona; and
¶ Departament de Química Orgànica, UB
1Correspondence: E-mail: xfernandez_busquets{at}ub.edu
SPECIFIC AIMS
Pathogenesis in Alzheimer’s disease (AD) is linked to the accumulation of the highly amyloidogenic self-associating amyloid ß1-42 peptide (Aß1-42). We have used atomic force microscopy (AFM) visualization on hydrophilic and hydrophobic surfaces in liquid environment to study in vitro the fibrillogenesis process of synthetic Aß1-42, with the aim of obtaining nanometric resolution of the main structures characteristic of the several steps of fibril formation from monomeric Aß1-42 to mature fibrils.
PRINCIPAL FINDINGS
1. Globular oligomers of Aß1-42 are the dominant species at early stages of aggregation and deposit best on hydrophilic surfaces
Aß1-42 was dissolved in phosphate buffer, pH 7.4, to a final concentration of 10 µM and immediately visualized in liquid medium on mica substrate by tapping mode AFM. At this initial point of incubation (t0), the sample contained homogeneously distributed globular aggregates with a diameter of 
5 nm. When imaged on a hydrophobic substrate such as graphite, Aß1-42 forms a layer 1 to 2 nm in height. Another aliquot of the same sample was analyzed by Western blot after electrophoresis in SDS-containing polyacrylamide gels. The band with highest electrophoretic mobility corresponded to a molecular mass roughly coincident with that of the Aß1-42 monomer (4.5 kDa). Three minor bands of lower electrophoretic mobility were detected, with apparent approximate masses of 7, 16, and 20 kDa that most likely correspond, respectively, to the dimer, trimer, and tetramer species of Aß1-42 that have resisted dissociation by SDS. The most abundant species detected at t0 in immunoblots was a wide band with an apparent mass of 60 kDa, which probably corresponds to larger heterogeneous SDS-resistant oligomers.
2. Ribbon-shaped Aß1-42 protofibrils deposit best on hydrophobic surfaces and do not grow longer than 100 nm
The next step in the process of Aß aggregation is the appearance of protofibrils, the first fibrillar species. Aß1-42 samples incubated for 24 h and deposited on freshly cleaved mica or graphite immediately before AFM imaging in liquid revealed the presence of ribbon-shaped protofibrils 1.5 nm high and 5.5 nm wide (see below), with an average length of 64.4 nm and seldom exceeding 100 nm. Protofibrils are visualized in much greater numbers when deposited on graphite, suggesting they have a hydrophobic surface and therefore settle down more efficiently on hydrophobic graphite rather than on hydrophilic mica. Protofibrils were observed to align in parallel, forming bundles that in turn can assemble end-to-end to form fibrils.
3. Most Aß1-42 fibrils exhibit a nodular structure with 100 nm periodicity
Abundant fibrils with a width of 11.4 nm and up to some microns long are observed in Aß1-42 preparations after 2 days of incubation (Fig. 1
A). Although protofibril bundle formation might be the initial step for fibril assembly, it is likely that individual protofibrils can also be incorporated into existing fibrils, as suggested by the observation of contacts between protofibril ends and fibrils (Fig. 1B
). We have identified two main types of Aß1-42 fibrils according to their contour: smooth and nodular (Fig. 1C
), the latter being 10-fold more abundant than the former. Nodular fibrils (Fig. 1C
, 1D
) have regularly spaced constrictions every 93.5 nm (Fig. 1E
) that are not present on smooth fibrils (Fig. 1F
). Smooth fibrils have a height of 5 nm, whereas the height of nodular fibrils varies between 11 nm in the center of the nodules and 5 nm at internodal points. A higher resolution scanning of the nodular fibril from Fig. 1C
reveals a grooved surface (Fig. 1D
) that delineates six elongated structures within each nodule (Fig. 1G
). Based on images of intermediate species, we deduce that the elongated structures are protofibrils, which have a width of 5.5 nm.
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4. Aß1-42 fibrillogenesis generates helical structures with a period of 100 nm
Left-handed helical structures formed by two or more strands are frequently observed in our in vitro preparations visualized by AFM and transmission electron microscopy (TEM). Each helix strand observed by TEM was 3.7 nm across, and the whole structure ranged from this narrowest width to 7.6 nm at the widest point of the helix. The period of half a helix turn (i.e., the distance between two consecutive narrow spots of the helix) differed between individual fibrils, varying from 39.0 nm to 149.4 nm, with a mean value of 92.5 nm. Each helix strand has a width close to that of protofibrils, but it can reach a length of several microns, and we will thus refer to each strand as protofilament.
5. Aß1-42 fibrils have structurally weak points every 100 nm
Figure 2
A shows an AFM image of partially assembled/disassembled fibrils, where the fibril nodules are hinted by the presence of apparently softer material that can be significantly pushed aside when applying greater forces with the AFM tip by increasing the working amplitude (Fig. 2B
). This manipulation of fibrils permitted to reveal the existence of an underlying protofilament helix with a period that coincides precisely with the length of nodules in the fibril. Eventually, AFM and TEM images show fibrils that have been fractured in sections (Fig. 2C-E
) with a mean fragment length of 107 nm, close to the measured periodicities of fibril nodules (93 nm), and of the helical repeat of intertwined protofilaments (93 nm) (Fig. 2F
). In these segmented fibrils the two protofilaments run parallel and do not present any twist (Fig. 2C
), and the protofilament sections strongly resemble protofibrils 100 nm long. Such fragmentation events suggest the existence of a structural weakness related to the joining points between the constituent 100 nm protofibril subunits.
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CONCLUSIONS
It is likely that the most abundant features revealed at the onset of Aß1-42 fibrillogenesis by AFM on mica (globular aggregates) and by Western blots (60 kDa band) correspond to the same molecular entity. This agrees with existing data indicating that, in a hydrophilic environment, Aß1-42 has a strong tendency to self-associate forming highly stable oligomeric structures termed ADDLs (amyloid-derived diffusible ligands), which may be the key effectors of cytotoxicity in AD. Small ADDLs include associations of four to six Aß1-42 units that have been described to form in the membrane of neurons the calcium-permeable channels that are suspected to be one of the causes of cell death associated with Aß. As an alternative mechanism recently described, ADDLs between 10 and 100 kDa have been shown to bind to certain synapses. The ensuing functional disruption may provide a molecular basis for synapse failure in memory loss.
The protofibril dimensions deduced from our AFM images (ribbon-like structures 1.5 nm high and 5.5 nm wide) are similar to those reported in previous studies of Aß1-42 fibrillogenesis. In these models, protofibrils are proposed to be single ß-sheets formed by a stacking of the peptide in a hairpin conformation perpendicular to the protofibril axis (Fig. 3
A), with a spacing of 5 Å between consecutive hydrogen bond-linked ß-strands. The mean length of isolated protofibrils is 64 nm and they seldom grow longer than 100 nm. This observation could be explained by a selective channeling of longer protofibrils approaching 100 nm toward their incorporation into bundles (Fig. 3Biii
), as suggested by the left-skewed histogram in Fig. 2F
corresponding to the distribution of protofibril lengths. Protofibril association is likely driven by the thermodynamically unfavorable exposure of the constituent hydrophobic ß-sheets to the aqueous environment. We have identified three other entities in the Aß1-42 fibrillogenetic pathway that share a 100 nm structural unit, namely, fibril nodules, helical protofilaments, and fibril fragments. Taken together, our data are consistent with the existence of a 100 nm long protofibril species with a role as short-lived assembly intermediate whose elongation rate is slower than its rate of association to form fibrils. Protofibril bundles stack end-to-end (Fig. 3Biv
) and appear to be capable of undergoing internal rearrangements leading to the generation of nodular fibrils (Fig. 3Bv
) and of a helical structure (Fig. 3Bvi
) that might play an important role in fibrillogenesis. We have observed a variety of helical periods that probably correspond to different points of coiling in the dynamic process leading from protofibrils to protofilaments and fibrils. Conformational changes experienced by protofibrils turning into protofilaments could be responsible for the different dimensions of isolated protofibrils (short ribbon-like structures 5.5 nm wide and 1.5 nm high) and of protofilaments (long cylinders 4 nm in diameter). Although Aß1-42 and a number of other amyloidogenic proteins and peptides are not sequence-related, they form twisted protofilaments and nodular fibrils remarkably similar in periodicity, dimensions, and number of constituent subunits. This is in agreement with the current view that, despite the different nature of precursor proteins and peptides, amyloid fibrils represent a structural superfamily and share a common protofilament structure.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3137fje;
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