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. 2011 Dec 18;19(1):79-83.
doi: 10.1038/nsmb.2191.

The Extracellular Chaperone Clusterin Sequesters Oligomeric Forms of the amyloid-β(1-40) Peptide

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The Extracellular Chaperone Clusterin Sequesters Oligomeric Forms of the amyloid-β(1-40) Peptide

Priyanka Narayan et al. Nat Struct Mol Biol. .
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Abstract

In recent genome-wide association studies, the extracellular chaperone protein, clusterin, has been identified as a newly-discovered risk factor in Alzheimer's disease. We have examined the interactions between human clusterin and the Alzheimer's disease-associated amyloid-β(1-40) peptide (Aβ(1-40)), which is prone to aggregate into an ensemble of oligomeric intermediates implicated in both the proliferation of amyloid fibrils and in neuronal toxicity. Using highly sensitive single-molecule fluorescence methods, we have found that Aβ(1-40) forms a heterogeneous distribution of small oligomers (from dimers to 50-mers), all of which interact with clusterin to form long-lived, stable complexes. Consequently, clusterin is able to influence both the aggregation and disaggregation of Aβ(1-40) by sequestration of the Aβ oligomers. These results not only elucidate the protective role of clusterin but also provide a molecular basis for the genetic link between clusterin and Alzheimer's disease.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Bulk and single molecule studies of Aβ1–40. (a) Appearance and disappearance of species populated during the aggregation of Aβ1–40 ( 2 µM at 37 °C with agitation). Fibril formation monitored by thioflavin T (ThT) fluorescence (top). The inset (top) is a transmission electron microscopy (TEM) image of the fibrils present after 24 h of incubation (scale bar is 200 nm). Concentration of soluble oligomers (dimers–50mers) (middle), and concentration of monomeric species (bottom) are both tracked using cTCCD. The data are averaged from multiple experimental repetitions (2 μM Aβ1–40, n=3, error bars are s.e.m.). (b) A representative distribution of apparent sizes of oligomers formed during Aβ1–40 aggregation and disaggregation (error bars are s.d.). Insets are zoomed into regions of dimers–15-mers and 16-mers–50-mers to provide greater detail. (c) A comparison of the distributions of apparent oligomer sizes during aggregation and disaggregation experiments (2 μM Aβ1–40 aggregation, n=3; disaggregation, n=12; 10–30 nM Aβ1–40 aggregation, n=4; error bars are s.e.m.). (d) Time dependence of the concentration of soluble species released from a pellet of fibrils (n=12, error bars are s.e.m.).
Figure 2
Figure 2
The effects of clusterin on the aggregation of Aβ1–40. (a) Fraction of oligomers detected in solution during the aggregation of Aβ1–40 with and without clusterin (Aβ1–40 and clusterin are both at a concentration of 600 nM, n=3, error bars are s.e.m.). (b) TIRFM image of the species present after 24 h of aggregation of a 2 μM solution of Aβ1–40 without clusterin (left). TIRFM image of a 2 μM solution of Aβ1–40 after 24 h of aggregation but with 2 µM clusterin added 4 h after the start of the reaction, during the fibril growth phase (right). An approximately 50% reduction in the average size of species present is observed in the presence of clusterin (from 1400 ± 200 nm without clusterin to 780 ± 60 nm with clusterin, s.e.m., P-value is 0.01, two-sample independent, two-tailed, t-test). Scale bars are 5 µm. (c) Fractions of species formed during the aggregation of a 2 μM solution that are oligomeric and that are in Aβ:clusterin complexes. (n=3, error bars are s.e.m.). (d) Proportion of Aβ:clusterin complexes persisting at 10–20 nM (total peptide concentration) at 21 °C. Complexes were formed between clusterin and oligomers from both aggregation and disaggregation reactions. For both traces, n=3, error bars are s.e.m. There is no statistically significant change in the proportion of complexes with oligomers formed during either the disaggregation experiment (P-value of 0.77, ANOVA single-factor) or the aggregation experiment (P-value of 0.99, ANOVA single-factor).
Figure 3
Figure 3
The effects of clusterin on the disaggregation of Aβ1–40 fibrils. (a) Distributions of apparent sizes of oligomers formed during aggregation and disaggregation reactions with and without clusterin. (Aggregation without clusterin, n=2, error bars are range; aggregation with clusterin, n=3; disaggregation without clusterin, n=10; disaggregation with clusterin, n=3; error bars are s.e.m.). (b) Time dependence of the release of soluble species during the disaggregation experiments in the presence and absence of clusterin (top), increased oligomer concentration in the presence of clusterin in the concentration plateau region (significant with a P-value of 0.002) (bottom left) and decreased monomer concentration in the presence of clusterin, in the concentration plateau region (significant with a P-value of 0.0003) (bottom right). Both correlations were analyzed using a two-sample independent, two tailed t-test, n=8, and error bars are s.e.m. (c) TIRFM imaging of HiLyte488Fluor-labeled Aβ1–40 fibrils incubated overnight at room temperature with AlexaFluor647-labeled clusterin. Aβ1–40 fluorescence only (left), clusterin fluorescence only (middle), and colocalization of the two species (right). Scale bars are 5 μm.

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