N-terminal domain of myelin basic protein inhibits amyloid beta-protein fibril assembly

Abstract

Accumulation of amyloid β-protein (Aβ) into brain parenchymal plaques and the cerebral vasculature is a pathological feature of Alzheimer disease and related disorders. Aβ peptides readily form β-sheet-containing oligomers and fibrils. Previously, we reported a strong interaction between myelin basic protein (MBP) and Aβ peptides that resulted in potent inhibition of fibril assembly (Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2007) J. Biol. Chem. 282, 9952-9961; Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2009) Biochemistry 48, 4720-4727). MBP is recognized as a highly post-translationally modified protein. In the present study, we demonstrate that human MBP purified from either brain or a bacterial recombinant expression system comparably bound to Aβ and inhibited Aβ fibril assembly indicating that post-translational modifications are not required for this activity. We also show that purified mouse brain MBP and recombinantly expressed mouse MBP similarly inhibited Aβ fibril formation. Through a combination of biochemical and ultrastructural techniques, we demonstrate that the binding site for Aβ is located in the N-terminal 64 amino acids of MBP and that a stable peptide (MBP1) comprising these residues was sufficient to inhibit Aβ fibrillogenesis. Under conditions comparable with those used for Aβ, the fibrillar assembly of amylin, another amyloidogenic peptide, was not inhibited by MBP1, although MBP1 still bound to it. This observation suggests that the potent inhibitory effect of MBP on fibril formation is not general to amyloidogenic peptides. Finally, MBP1 could prevent the cytotoxic effects of Aβ in primary cortical neurons. Our findings suggest that inhibition of Aβ fibril assembly by MBP, mediated through its N-terminal domain, could play a role in influencing amyloid formation in Alzheimer disease brain and corresponding mouse models.

FIGURE 1.

Inhibition of Aβ fibril formation by purified human brain MBP and recombinant His-tagged human MBP. A, MBP was either purified from human white matter (lane 1) or recombinantly expressed in bacteria as a His-tagged protein (lane 2) and assessed by SDS-PAGE. B, interaction of Aβ40DI with human brain MBP (hMBP) or His-tagged human MBP was analyzed by solid phase binding assay. The data shown are the mean ± S.D. of triplicate determinations. C, inhibition of Aβ40DI (12.5 μm) fibrillogenesis by either purified human brain MBP (1.56 μm) or purified His-tagged human MBP (1.56 μm) as assessed by thioflavin T binding and fluorescence. Aβ40DI alone, ♦; Aβ40DI + human brain MBP, ■; Aβ40DI + His-tagged human MBP, ▴. The data shown are the mean ± S.D. of triplicate determinations. D, TEM analysis of Aβ40DI with and without human brain MBP or His-tagged human MBP. Both forms of human MBP inhibit Aβ40DI fibril formation. Scale bars, 100 nm. A.U., absorbance units.

FIGURE 2.

Inhibition of Aβ fibril formation by purified mouse brain and recombinant His-tagged mouse MBP. A, MBP was either purified from mouse brain (lane 1) or recombinantly expressed in bacteria as a His-tagged protein (lane 2) and assessed by SDS-PAGE. B, interaction of Aβ40DI with mouse brain MBP (mMBP) or His-tagged mouse MBP was analyzed by solid phase binding assay. The data shown are the mean ± S.D. of triplicate determinations. C, inhibition of Aβ40DI (12.5 μm) by purified mouse brain MBP (1.56 μm) or purified His-tagged mouse MBP (1.56 μm) as assessed by thioflavin T binding and fluorescence. Aβ40DI alone, ♦; Aβ40DI + mouse MBP, ■; Aβ40DI + His-tagged mouse MBP, ▴. The data shown are the mean ± S.D. of triplicate determinations. D, TEM analysis of Aβ40DI with and without mouse brain MBP or His-tagged mouse MBP. Both forms of mouse MBP inhibit Aβ40DI fibril formation. Scale bars, 100 nm. A.U., absorbance units.

FIGURE 3.

Sequence of recombinant MBP peptides. MBP was divided into four peptides for recombinant bacterial expression and purification. a.a., amino acids.

FIGURE 4.

Binding of MBP peptides to Aβ as assessed by SPR binding analysis. A, each of the MBP peptides was expressed in bacteria, purified, and assessed by SDS-PAGE. B, each of the purified MBP peptides (50 nm) was passed over immobilized Aβ40DI ligand. Representative sensorgrams were base line-corrected and plotted as overlays. Binding is identified by an increase in response during injection (Inj.) (association) followed by a gradual decrease in response (dissociation). MBP1, black line; MBP2, green line; MBP3, blue line; MBP4, red line). Only MBP1 demonstrated binding to the Aβ40DI ligand.

FIGURE 5.

Thioflavin T analysis of inhibition of Aβ fibril formation by purified MBP peptides. Aβ40DI was treated with hexafluoroisopropanol, resuspended to a concentration of 2.5 mm in DMSO, and then diluted to a concentration of 12.5 μm in PBS in the absence (□) or presence of 1.56 μm MBP peptides. Aβ40DI + MBP1, ●; Aβ40DI + MBP2, ♦; Aβ40DI + MBP3, ■; Aβ40DI + MBP4, ▴). At specific time points aliquots were collected from each sample and assayed by thioflavin T binding and fluorescence to determine fibrillar assembly. MBP1 is as capable as intact MBP at inhibiting Aβ40DI fibrillogenesis. The data shown are the mean ± S.D. of triplicate samples. A.U., absorbance units.

FIGURE 6.

MBP1 binds to and inhibits Aβ peptide fibril formation. A, interaction of Aβ42WT and Aβ40DI with MBP or MBP1 was analyzed by solid phase binding assay. The data shown are the mean ± S.D. of triplicate determinations. B–D, 100 μm Aβ42WT was incubated for 24 h in the absence or presence of 12.5 μm MBP1 or MBP. Samples were imaged by TEM (scale bars, 100 nm). E–J, 12.5 μm Aβ40DI was incubated for 6 h in the absence or presence of 1.56 μm MBP1 or MBP. Samples were imaged by TEM (scale bars in E–G, 100 nm) and by AFM (scale bars in H–J, 100 nm). Both MBP1 and MBP are capable of binding and inhibiting Aβ42WT or Aβ40DI fibrillogenesis.

FIGURE 7.

MBP binds to amyloidogenic IAPP but does not inhibit its fibril formation. A, interaction of Aβ40DI and IAPP with MBP and MBP1 was analyzed by solid phase binding assay. The data shown are the mean ± S.D. of triplicate determinations. B, IAPP peptide was treated with hexafluoroisopropanol, resuspended to a concentration of 2.5 mm in DMSO, and then diluted to a concentration of 3.13 μm in PBS in the absence (■) or presence (♦) of 0.391 μm MBP1. At specific time points, aliquots were collected from each sample and assayed by thioflavin T binding and fluorescence to determine fibrillar assembly. The data shown are the mean ± S.D. of triplicate samples. C, at the conclusion of the thioflavin T assay, samples were imaged by TEM. Scale bars, 500 nm. MBP1 does not inhibit the fibrillogenesis of IAPP. hMBP, human MBP.

FIGURE 8.

MBP1 protects neurons from Aβ42WT toxicity. In the cell viability assay, primary rat cortical neurons were treated with 10 μm Aβ42WT in the absence of presence of 1.25 μm MBP1 for 18 h. Cell viability was determined using the MTT assay. The data shown are the mean ± S.D. of six independent samples. *, p < 0.001. con, control.