Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer's disease

Abstract

In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinson's disease and Alzheimer's disease. As a consequence, research efforts are being directed away from traditional targets, such as amyloid plaques, and towards characterization of early oligomer states. Here we present a new analysis method, ion mobility coupled with mass spectrometry, for this challenging problem, which allows determination of in vitro oligomer distributions and the qualitative structure of each of the aggregates. We applied these methods to a number of the amyloid-β protein isoforms of Aβ40 and Aβ42 and showed that their oligomer-size distributions are very different. Our results are consistent with previous observations that Aβ40 and Aβ42 self-assemble via different pathways and provide a candidate in the Aβ42 dodecamer for the primary toxic species in Alzheimer's disease.

Figure 1. Negative-ion mass spectrum and ATDs of Aβ40

a, Mass spectrum from a 30 μM solution at pH 7.4 labelled with z/n values (z = charge, n = oligomer order). The insets show high-resolution ¹³C isotope patterns. The spacing of 0.33 for the z/n = −3 peak indicates that it is a monomer. The more complex pattern for z/n = −2 indicates that both monomer and dimer are present (dimer: z = −4, n = 2). b,c, ATDs at high and low injection energies (IE) for z/n = −2 (b) and z/n = −5/2 (c). The vertical dotted lines show the expected average experimental arrival times of the dimer and tetramer. M = monomer, D = dimer, Te = tetramer (see text).

Figure 2. Arrival time distributions

a–f, ATDs for z/n = −5/2 Aβ42 (a–c) and Aβ40 (d–f), showing the wild types (a,d), the alloforms Pro¹⁹ (b,e) and Met³⁵(O) (c,f). The hexamer and dodecamer are only observed for Aβ42 wild type and are thus implicated in the increased toxicity for this alloform. The distributions in a and b are from Bernstein et al. D = dimer, Te = tetramer, H = hexamer, Do = dodecamer.

Figure 3. Aβ42 oligomer distributions

a, The ATDs of z/n = −5/2 with structural designations in place. b, A plausible mechanism for forming the oligomer distribution given by the ATD in a.

Figure 4. The normalized cross-sections for the tetramers

a, Relative sizes of all tetramers reported herein (shown to right of vertical scale). The vertical scale is the relative cross-section of the model tetramers normalized to the linear structure. The broad arrows on the vertical axis show the spread of values for the modelled structures. Aβ42 is a much more open tetramer than the other alloforms. b, The structures of the Aβ42 and Aβ40 tetramers taken from their location on the relative cross-section scale given in a, assuming the angle between dimer components is the determining parameter.

Figure 5. Mechanism of oligomerization and eventual fibril formation for Aβ42 and for Aβ40

For Aβ40 the key structure is the tetramer that resists further monomer or dimer addition. In Aβ42 an ‘open’ tetramer promotes the formation of the planar hexamer (paranucleus) and the stacked dodecamer, which resists further reaction. For Aβ40 the tetramer apparently eventually forms fibrils (observed by others), but these were not observed in our experiments. For Aβ42 a rate-limiting slow α- to β-sheet transformation may occur for the dodecamer, but this was not explicitly observed in our experiments. Fibril formation was indirectly observed through macroscopic clogging of the spray tips used for Aβ42.