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. 2013 May;27(5):1847-58.
doi: 10.1096/fj.12-222588. Epub 2013 Jan 18.

The cellular prion protein traps Alzheimer's Aβ in an oligomeric form and disassembles amyloid fibers

Nadine D Younan  1 , Claire J Sarell, Paul Davies, David R Brown, John H Viles
Affiliations

The cellular prion protein traps Alzheimer's Aβ in an oligomeric form and disassembles amyloid fibers

Nadine D Younan et al. FASEB J. 2013 May.

Abstract

There is now strong evidence to show that the presence of the cellular prion protein (PrP(C)) mediates amyloid-β (Aβ) neurotoxicity in Alzheimer's disease (AD). Here, we probe the molecular details of the interaction between PrP(C) and Aβ and discover that substoichiometric amounts of PrP(C), as little as 1/20, relative to Aβ will strongly inhibit amyloid fibril formation. This effect is specific to the unstructured N-terminal domain of PrP(C). Electron microscopy indicates PrP(C) is able to trap Aβ in an oligomeric form. Unlike fibers, this oligomeric Aβ contains antiparallel β sheet and binds to a oligomer specific conformational antibody. Our NMR studies show that a specific region of PrP(C), notably residues 95-113, binds to Aβ oligomers, but only once Aβ misfolds. The ability of PrP(C) to trap and concentrate Aβ in an oligomeric form and disassemble mature fibers suggests a mechanism by which PrP(C) might confer Aβ toxicity in AD, as oligomers are thought to be the toxic form of Aβ. Identification of a specific recognition site on PrP(C) that traps Aβ in an oligomeric form is potentially a therapeutic target for the treatment of Alzheimer's disease.

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Figures

Figure 1.
Figure 1.
PrPC inhibits Aβ40 fiber formation. Kinetics of Aβ40 fiber formation was monitored by fluorescence upon ThT binding to amyloid. Aβ40 alone (A), and in the presence of 1 mol eq (B), 0.1 mol eq (C), 0.05 mol eq (D), 0.025 mol eq (E), and 0.01 mol eq (F) of PrP(23–231). Aβ40 monomer (10 μM) was incubated at pH 7.4 in HEPES buffer (30 mM) and NaCl (160 mM) at 30°C with intermittent agitation. As little as 500 nM of PrP(23–231) completely inhibits Aβ40 fiber formation over 250 h.
Figure 2.
Figure 2.
PrPC inhibits Aβ42 fiber formation. Kinetics of Aβ42 (5 μM) fiber formation was monitored by fluorescence upon ThT binding to amyloid. Aβ42 alone (A), and in presence of 1 mol eq (B), 0.1 mol eq (C), 0.05 mol eq (D), and 0.02 mol eq (E) of PrP(23–231). Aβ42 monomer (5 μM) was incubated at pH 7.4 in HEPES buffer and 160 mM NaCl at 30°C with intermittent agitation. As little as 1:20 PrP(23–231) inhibits Aβ42 fiber formation over 250 h.
Figure 3.
Figure 3.
Aβ fiber formation in the presence of PrPC fragments. Kinetics of Aβ40 (10 μM) fiber formation was monitored by fluorescence upon ThT binding to amyloid. Aβ40 alone (A), and in presence of 1 mol eq of PrP(113–231) (B), 0.1 mol eq of PrP(23–126) (C), 0.1 mol eq of PrP(58–91) (D), 0.1 mol eq of PrP(91–115) (E), and 1 mol eq of PrP(91–115) (F). Aβ40 was incubated at pH 7.4 in 30 mM HEPES buffer and 160 mM NaCl at 30°C with intermittent agitation.
Figure 4.
Figure 4.
PrPC induced Aβ fiber disassembly. Mature Aβ40 fibers alone (gray) are formed over a 234-h period, after which 1 mol eq of PrP(23–231) is added to 6 of the reaction wells (black). Aβ40 (10 μM) samples were incubated at pH 7.4 in 30 mM HEPES buffer and 160 mM NaCl at 30°C with intermittent agitation. Addition of PrPC caused a 40% reduction in ThT fluorescence within 90 min.
Figure 5.
Figure 5.
15N HSQC NMR of the PrP(23–231) binding to Aβ40 oligomer. A, B) Selected regions of 2D 15N-1H HSQC of PrP(23–231) alone (black) and PrP(23–231) with 1 mol eq of Aβ40 after 40 h incubation (red). Amide resonances that show a marked loss of signal are labeled in blue. C) Peak intensity plotted against time; 6 new peaks are observed after 20 h (various shades of red), as well as reductions in the intensity of A113 and Q98, while S131 and T187 remain unaffected over 40 h. Spectra obtained at 30°C in 50 mM phosphate buffer (pH 6.5) and 50 μM PrPC and Aβ.
Figure 6.
Figure 6.
TEM of Aβ in the presence of PrPC. Negative-stain TEM images of Aβ40 fibers alone (A), Aβ incubated with PrP(23–231) (B–E), and PrPC added to mature Aβ40 fibers (F). Aβ40 (10 μM) samples were incubated at pH 7.4 in 30 mM HEPES and 160 mM NaCl at 30°C with intermittent agitation for 300 h. TEM grids were negatively stained using phosphotungstic acid. Only Aβ oligomers are observed where Aβ is incubated with PrPC (0.1 mol eq).
Figure 7.
Figure 7.
SEC of Aβ40 oligomer in the presence of PrPC. Aβ40 was incubated with PrP(23–231) (A) and PrP(23–126) (B). Traces indicate Aβ40 only (top), Aβ40 with 0.5 μM PrP (middle), and Aβ40 with 2 μM PrP (bottom). Labels I and II indicate oligomers I and II, the complexes formed at ∼60 and ∼100 kDa, respectively. Aβ40 (10 μM) samples with and without PrP were incubated at pH 7.4 in 30 mM HEPES and 160 mM NaCl at 30°C with agitation for 250 h. SEC was carried out at 4 °C and pH 7.4, using a Superdex 200 column.
Figure 8.
Figure 8.
Aβ oligomer antibody binding dot-blot assay. Samples were dotted on a membrane and examined using antibody A11, which is sensitive to oligomers but not to fibers or monomers. A) Aβ42 essentially monomeric. B) Aβ42 (30 μM) incubated with PrP(23–231) (10 μM). C) Aβ42 fibers. D) Disassembled Aβ42 fibers with 1.5 mol eq of PrP(23–231). E) PrP(23–126) (10 μM) only. F) Aβ40 with 0.1 mol eq of PrP(23–126). G) PrP(23–231) (10 μM) only.
Figure 9.
Figure 9.
IR spectra of Aβ oligomers in the presence of PrPC. A) Structural characterization using IR amide-I band of Aβ40 mature fibers (dashed gray) and Aβ42 mature fibers (solid black). B) Aβ40 monomer incubated with 0.1 mol eq of PrP(23–231) (solid black) or PrP(23–231) alone (dashed gray). C) Aβ40 with PrPC oligomers eluted by SEC. D) Aβ40 mature fibers with 1 mol eq of PrP(23–231) (solid black), PrP(23–231) alone (dashed gray), and difference spectra (dotted). Aβ40 monomer (10 μM) was incubated at pH 7.4 in 30 mM HEPES buffer and 160 mM NaCl at 30°C. Vertical dashed gray lines highlight 1695 and 1633 cm−1. Aβ40 oligomer formed in the presence of PrPC shows an increase in 1695 cm−1 amide-I band.
Figure 10.
Figure 10.
PrPC stabilizes the oligomeric form of Aβ. Binding of PrPC stops the transition from antiparallel to parallel sheet necessary for a transition from oligomer to fiber. PrPC may stop the rotation of the β-sheet pairing from intra- to intermolecular. The energy barrier to go from antiparallel to parallel arrangement of β sheet is likely to be very large. It is therefore more likely that antiparallel oligomeric arrangements largely disassemble to form the in-register parallel sheets formed in fibers. Whether the transition from antiparallel oligomer to fibers is on or off pathway, it is clear that the binding of PrPC stabilizes the oligomeric form.
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